Please note: By no means consider this a comprehensive listing of cloning related literature, but merely a select compendium of studies which I believe illustrate important findings applicable to mammalian cloning. Please pay special attention to the comments! I am working to add more detail from the reviewed literature, however this is a very time-consuming process. Eventually I will get around to fleshing out the discussion and protocol pages, largely on the basis of what I site on this page.
If you have additional applicable references or observations to offer, please email me!If you are interested in discussing any of these or related articles please do so at the Human Cloning Foundation's discussion Board: "The Science of Cloning" . If you would like to maintain a private discussion, email me!
Hum Reprod 2000 Jun;15(6):1396-9
Embryos with high implantation potential after intracytoplasmic sperm injection can be recognized by a simple, non-invasive examination of pronuclear morphology.
Tesarik J, Junca AM, Hazout A, Aubriot FX, Nathan C, Cohen-Bacrie P, Dumont-Hassan MHum Reprod Update 1998 Mar-Apr;4(2):121-34
Relationships between the developmental potential of human in-vitro fertilization embryos and features describing the embryo, oocyte and follicle.
Saith RR, Srinivasan A, Michie D, Sargent ILA Novel Methodology Allows Detection of Every Chromosome in Single Blastomeres from Human Preimplantation Embryos and Reveals Aneuploidy, Mosaicism and Uniformly Normal Embryos
D. Wells and J.D.A. Delhanty Dept. Obs. & Gynae, University College London, Chenies Mews, London WC1E 6HX, UK
Nature 1997 Feb 27;385(6619):810-3 Published erratum appears in Nature 1997 Mar 13;386(6621):200
Viable offspring derived from fetal and adult mammalian cells.
Wilmut I, Schnieke AE, McWhir J, Kind AJ, Campbell KHWakayama T, Perry AC, Zuccotti M, Johnson KR, Yanagimachi R
Full-term development of mice from enucleated oocytes injected with cumulus cell nuclei
Nature 1998 Jul 23;394(6691):369-74
Nat Genet 1999 Jun;22(2):127-8
Cloning of male mice from adult tail-tip cells.
Wakayama T, Yanagimachi RCLONING Volume 1, Number 3, 1999 Mary Ann Liebert, Inc.
Mammalian Leukocytes Contain All the Genetic Information Necessary for the Development of a New Individual
CESARE GALLI,1 ROBERTO DUCHI,1 ROBERT M. MOOR,2 and GIOVANNA LAZZARI 1Science 1998 Dec 11;282(5396):2095-8
Eight calves cloned from somatic cells of a single adult.
Kato Y, Tani T, Sotomaru Y, Kurokawa K, Kato J, Doguchi H, Yasue H, Tsunoda YJ Reprod Fertil 2000 Nov;120(2):231-237
Cloning of calves from various somatic cell types of male and female adult, newborn and fetal cows.
Kato Y, Tani T, Tsunoda YScience 1998 May 22;280(5367):1256-8
Cloned transgenic calves produced from non quiescent fetal fibroblasts.
Cibelli JB, Stice SL, Golueke PJ, Kane JJ, Jerry J, Blackwell C, Ponce de Leon FA, Robl JMProc Natl Acad Sci U S A 2000 Feb 1;97(3):990-5
Six cloned calves produced from adult fibroblast cells after long-term culture.
Kubota C, Yamakuchi H, Todoroki J, Mizoshita K, Tabara N, Barber M, Yang XReprod Fertil Dev 1998;10(4):369-78
Adult somatic cell nuclear transfer is used to preserve the last surviving cow of the Enderby Island cattle breed.
Wells DN, Misica PM, Tervit HR, Vivanco WHBiol Reprod 1999 Apr;60(4):996-1005
Production of cloned calves following nuclear transfer with cultured adult mural granulosa cells.
Wells DN, Misica PM, Tervit HR.J Reprod Fertil 1999 Mar;115(2):325-31
Effects of serum starvation and re-cloning on the efficiency of nuclear transfer using bovine fetal fibroblasts.
Zakhartchenko V, Durcova-Hills G, Stojkovic M, Schernthaner W, Prelle K, Steinborn R, Muller M, Brem G, Wolf E.Nature 2000 Sep 7;407(6800):86-90
Cloned pigs produced by nuclear transfer from adult somatic cells.
Polejaeva IA, Chen SH, Vaught TD, Page RL, Mullins J, Ball S, Dai Y, Boone J, Walker S, Ayares DL, Colman A, Campbell KH
Cloning Volume: 1 Number: 1 Page: 63 -- 69 RETRIEVE FREE FULL TEXT ARTICLE
Reprogramming of Fibroblast Nuclei after Transfer into Bovine Oocytes
Paul A. De Sousa; Quinton Winger; Jonathan R. Hill; Karen Jones; Andrew J. Watson; Mark E. Westhusin
DOI: 10.1089/15204559950020102 Publisher: Mary Ann Liebert, Inc.Genes Dev 1999 Sep 15;13(18):2339-52 LINK TO FULL TEXT ARTICLE
ATP-dependent remodeling and acetylation as regulators of chromatin fluidity.
Kingston RE, Narlikar GJScience 2000 Sep 29;289(5488):2360-2
Active remodeling of somatic nuclei in egg cytoplasm by the nucleosomal ATPase ISWI.
Kikyo N, Wade PA, Guschin D, Ge H, Wolffe APProc Natl Acad Sci U S A 1999 May 25;96(11):5894-6
DNA demethylation.
Wolffe AP, Jones PL, Wade PA
Crit Rev Eukaryot Gene Expr 2000;10(1):1-12
Understanding "active" chromatin: a historical perspective of chromatin remodeling.
Krebs JE, Peterson CLChem Biol 1997 Dec;4(12):885-8
Protein acetylation: more than chromatin modification to regulate transcription.
Bayle JH, Crabtree GRCell Mol Life Sci 1998 Jan;54(1):6-20
Linking histone acetylation to transcriptional regulation.
Mizzen CA, Allis CD
Semin Perinatol 2000 Feb;24(1):46-50
The role of nitric oxide in apoptosis.
Li J, Billiar TR
GeneralRes Exp Med (Berl) 1988;188(5):391-6
Effects of pantothenic acid on fibroblastic cell cultures.
Lacroix B, Didier E, Grenier JF
Hematopoeitic Stem and Progenitor CellsThiemann FT, Moore KA, Smogorzewska EM, Lemischka IR, Crooks GM
The murine stromal cell line AFT024 acts specifically on human CD34+CD38- progenitors to maintain primitive function and immunophenotype in vitro.
Exp Hematol 1998 Jul;26(7):612-9
Lansdorp PM
Stem cell biology for the transfusionist
Vox Sang 1998;74 Suppl 2:91-4Moore KA , Ema H , Lemischka IR
In vitro maintenance of highly purified, transplantable hematopoietic stem cells.
Blood 1997 Jun 15;89(12):4337-47Aiuti A, Friedrich C, Sieff CA, Gutierrez-Ramos JC
Identification of distinct elements of the stromal microenvironment that control human hematopoietic stem/progenitor cell growth and differentiation.
Exp Hematol 1998 Feb;26(2):143-157Epidermal Stem Cells
Bickenbach JR, Chism E
Selection and extended growth of murine epidermal stem cells in culture.
Exp Cell Res 1998 Oct 10;244(1):184-95
J Assist Reprod Genet 1999 Apr;16(4):216-20
Calcium signaling in human preimplantation development: a review.
Tesarik JNature 2000 Aug 10;406(6796):633-6
NO is necessary and sufficient for egg activation at fertilization.
Kuo RC, Baxter GT, Thompson SH, Stricker SA, Patton C, Bonaventura J, Epel DBiochem J 1998 Nov 15;336 ( Pt 1):1-17
Arginine metabolism: nitric oxide and beyond.
Wu G, Morris SM Jr
Biol Reprod 1999 Jun;60(6):1483-7
Retinol administration to superovulated ewes improves in vitro embryonic viability.
Eberhardt DM, Will WA, Godkin JDFertil Steril 2001 Feb;75(2):348-353 View Full text Article Online ( Subscibers only)
The spindle observation and its relationship with fertilization after intracytoplasmic sperm injection in living human oocytes.
Wang W, Meng L, Hackett RJ, Odenbourg R, Keefe DL
Funahashi H, Cantley TC, Day BN.
Synchronization of meiosis in porcine oocytes by exposure to dibutyryl cyclic adenosine monophosphate improves developmental competence following in vitro fertilization.
Biol Reprod 1997 Jul;57(1):49-53Hum Reprod 2000 May;15(5):1149-54
Chemically and mechanically induced membrane fusion: non-activating methods for nuclear transfer in mature human oocytes.
Tesarik J, Nagy ZP, Mendoza C, Greco E
Theriogenology: January 1 2001 Vol.55 No.1, page 241
Culture in Microchannels Enhances In Vitro Embryonic Development of Preimplantation Mouse Embryos
S. Raty1, J.A. Davis1, D.J Beebe1,2 , S.L. Rodrireuz-Zas1, and M.B. Wheeler1
1University of Illinois at Urbana-Champaign, Urbana, IL, USA,2 University of Wsconsin-Madison, Madison,WI,USABiol Reprod 1999 Aug;61(2):541-7
Coenzyme Q(10) in submicron-sized dispersion improves development, hatching, cell proliferation, and adenosine triphosphate content of in vitro-produced bovine embryos.
Stojkovic M, Westesen K, Zakhartchenko V, Stojkovic P, Boxhammer K, Wolf EHum Reprod 2000 Jan;15(1):157-64
Culture of one-cell hamster embryos with water soluble vitamins: pantothenate stimulates blastocyst production.
McKiernan SH, Bavister BDTheriogenology 1998 Jan 1;49(1):83-102
Changes in requirements and utilization of nutrients during mammalian preimplantation embryo development and their significance in embryo culture.
Gardner DKMol Reprod Dev 2000 Sep;57(1):48-54
Potent and stage-specific action of glutathione on the development of goat early embryos in vitro.
Lee CS, Koo DB, Fang N, Lee Y, Shin ST, Park CS, Lee KKMol Reprod Dev 1999 Feb;52(2):149-57
Pyruvate prevents peroxide-induced injury of in vitro preimplantation bovine embryos.
Morales H, Tilquin P, Rees JF, Massip A, Dessy F, Van Langendonckt ASherbahn R, Frasor J, Radwanska E, Binor Z, Wood-Molo M, Hibner M, Mack S, Rawlins RG
Comparison of mouse embryo development in open and microdrop co-culture systems.
Hum Reprod 1996 Oct;11(10):2223-9Mutat Res 1997 Dec 12;396(1-2):113-27
Developmental toxicity induced during early stages of mammalian embryogenesis.
Rutledge JC
Hum Reprod 2000 Jun;15(6):1396-9
Embryos with high implantation potential after intracytoplasmic sperm injection can be recognized by a simple, non-invasive examination of pronuclear morphology.
Tesarik J, Junca AM, Hazout A, Aubriot FX, Nathan C, Cohen-Bacrie P, Dumont-Hassan MLaboratoire d'Eylau, 55 Rue Saint-Didier, 75116 Paris, France.
Embryos are conventionally selected for transfer based on the evaluation of the cleavage speed and extent of blastomere fragmentation. Here we examined whether the predictive value of these criteria, as indicators of the chance of embryo implantation, can be further potentiated by adding previously described criteria reflecting the regularity of pronuclear development. In a group of embryos selected for transfer in 380 fresh embryo transfer cycles according to the conventional criteria, the transfer of only those embryos that developed from zygotes judged normal at the pronuclear stage (pattern 0) gave significantly higher pregnancy (44.8%) and implantation (30.2%) rates compared with the pregnancy (22.1%; P < 0. 05) and implantation rates (11.2%; P < 0.001) for the transfers of only those embryos that developed from zygotes judged abnormal (non-pattern 0). The transfer of only one pattern 0 embryo was sufficient for the optimal chance of pregnancy (no differences in pregnancy rates after transfer of one, two or three pattern 0 embryos), whereas the transfer of two pattern 0 embryos mostly resulted in a twin pregnancy. The inclusion of the criteria based on pronuclear morphology can thus lead to the application of a single embryo transfer policy and optimize the selection of embryos for transfer and cryopreservation.
PMID: 10831576, UI: 20293246Comment: Pronuclear morphology is an important morphologic grading criterion for assessment of the zygote.
Hum Reprod Update 1998 Mar-Apr;4(2):121-34
Relationships between the developmental potential of human in-vitro fertilization embryos and features describing the embryo, oocyte and follicle.
Saith RR, Srinivasan A, Michie D, Sargent ILNuffield Department of Obstetrics and Gynaecology, John Radcliffe Hospital,
University of Oxford, UK.This paper investigates the relationship of features of in-vitro fertilization (IVF) embryos and the associated oocyte and follicle to the outcome of transfer. It differs from previous studies in including a range of features (n = 53) and in using class probability tree analysis. This is a non-parametric multivariate method which expresses relationships as simple rules of features characterizing the 'take home baby' and 'no take home baby' (negative pregnancy test) classes of embryo batches. Data were analysed retrospectively. Fifty-three (embryo, oocyte and follicular) features for each of the three embryos in the transferred batch were collected for 200 IVF patients. Each batch of three embryos was described by a representative value for each feature. The relationship between features and outcome of transfer was analysed. Only four of the 53 features were identified as predictive. However, an appropriate combination of these four (embryo grade, cell number, follicle size and follicular fluid volume) achieved satisfactory predictivities while offering a simplified and more quantitative basis than regularly used criteria. The key component of the composite embryo grading allotted by embryologists turned out to be cell number. The existing predictive use of follicular size was corroborated and an independent additional predictive contribution of follicular fluid volume was found. The study also suggests that the 49 remaining features have little additional predictive value.
PMID: 9683350
Comment: Cell Number is the most important criterion for embryo grading.
A Novel Methodology Allows Detection of Every Chromosome in Single Blastomeres from Human Preimplantation Embryos and Reveals Aneuploidy, Mosaicism and Uniformly Normal EmbryosD. Wells and J.D.A. Delhanty
Dept. Obs. & Gynae, University College London, Chenies Mews, London WC1E 6HX, UKObjectives: Conventional cytogenetic techniques have revealed high levels of chromosomal abnormality in human preimplantation embryos, but can only detect a small number of chromosomes in each cell. This has led some researchers to speculate that if a full analysis of all 23 chromosome pairs were possible it would reveal that all human embryos contain a proportion of abnormal cells. We have developed a novel technique to ascertain the true extent of chromosome abnormality in human embryos.
Design: Normally developing human preimplantation embryos (day 3) were disagreagated and each cell was subjected to cytogenetic analysis.
Materials and Methods: Sixty four isolated cells (blastomeres) from 12 embryos were subjected to whole genome amplification (WGA) followed by comparative genomic hybridization (CGH) to reveal the copy number of each chromosome.
Results: Eight out of 12 embryos were found to be mosaic, each containing more than one chromosomally distinct cell line. Two embryos were highly abnormal with apparent random or 'chaotic' segregation of chromosomes. Nine embryos contained a proportion of normal cells, but only 3 embryos displayed a balanced karyotype in all the cells assessed. The normal embryos could not be distinguished from aneuploid, mosaic or chaotic embryos on the basis of morphological criteria. In addition to loss and gain of whole chromosomes partial gains and losses indicating chromosome damage were also identified in two embryos.
Conclusions: We have developed a novel technique based on WGA and CGH, which for the first time allows the copy number of every chromosome to be assessed in the majority of cells from a preimplantation embryo. We have detected unusual forms of aneuploidy that are incompatible with postimplantation development, high levels of chromosomal mosaicism, non-mosaic aneuploidy and chromosome breakage. However our report also indicates that some embryos do have normal chromosome numbers in every cell. Such embryos may have a superior developmental potential, and their low frequency may explain the correspondingly low success rates of natural and assisted conception in humans. It is possible that detection and preferential transfer of chromosomally normal embryos could lead to an increase in pregnancy rate.
This study was supported by the Wellcome Trust (grants O46416 and O39938) and by the Medical Research Council (UK).
Comment: This was presented in abstract form only at the 2000 ASRM meeting. The study is significant in that it allows assessment at the chromosomal level, above and beyond that of morphological blastocyst grading, and allows for the identification of a relatively small subset of embryos with a perfectly normal complement of chromosomes ( only 3 of 12(25%) normal! ) It would appear that in this study entire embryos were disaggregated and each cell assessed.Perhaps about half of the blastomeres of a given embryo could be assessed following diaggregating of high morphological grade embryos. The remaining blastomeres of embryos that test normal could then be used in a second stage nuclear transfer, or establishment of an embryonic stem cell culture, with subsequent transfer following additional cell divisions under appropriate culture conditions. Second stage nuclear transfer alone has repeatedly demonstrated markedly increased rates and quality of blastocyst formation. In some studies, animals born of double SCNT procedures have been uniformly healthy. (Polaeva, et al)
Nature 1997 Feb 27;385(6619):810-3
Published erratum appears in Nature 1997 Mar 13;386(6621):200
Viable offspring derived from fetal and adult mammalian cells.
Wilmut I, Schnieke AE, McWhir J, Kind AJ, Campbell KH
Roslin Institute (Edinburgh), Roslin, Midlothian, UK. Ian.Wilmut@bbsrc.ac.uk
Fertilization of mammalian eggs is followed by successive cell divisions and progressive differentiation, first into the early embryo and subsequently into all of the cell types that make up the adult animal. Transfer of a single nucleus at a specific stage of development,to an enucleated unfertilized egg, provided an opportunity to investigate whether cellular differentiation to that stage involved irreversible genetic modification. The first offspring to develop from a differentiated cell were born after nuclear transfer from an embryo-derived cell line that had been induced to become quiescent. Using the same procedure, we now report the birth of live lambs from three new cell populations established from adult mammary gland, fetus and embryo. The fact that a lamb was derived from an adult cell confirms that differentiation of that cell did not involve the irreversible modification of genetic material required for development to term. The birth of lambs from differentiated fetal and adult cells also reinforces previous speculation that by inducing donor cells to become quiescent it will be possible to obtain normal development from a wide variety of differentiated cells.
PMID: 9039911, UI: 97192092Quotes & Notes:Pending
Comment: It is this paper which launched the entire controversy, it only represents an incremental step of many years of preceding research.
Nature 1998 Jul 23;394(6691):369-74
Full-term development of mice from enucleated oocytes injected with cumulus cell nuclei.
Wakayama T, Perry AC, Zuccotti M, Johnson KR, Yanagimachi RDepartment of Anatomy and Reproductive Biology, John A. Burns School of Medicine, University of Hawaii, Honolulu 96822, USA.
Until recently, fertilization was the only way to produce viable mammalian offspring, a process implicitly involving male and female gametes. However, techniques involving fusion of embryonic or fetal somatic cells with enucleated oocytes have become steadily more successful in generating cloned young. Dolly the sheep was produced by electrofusion of sheep mammary-derived cells with enucleated sheep oocytes. Here we investigate the factors governing embryonic development by introducing nuclei from somatic cells (Sertoli, neuronal and cumulus cells) taken from adult mice into enucleated mouse oocytes. We found that some enucleated oocytes receiving Sertoli or neuronal nuclei developed in vitro and implanted following transfer, but none developed beyond 8.5 days post coitum; however, a high percentage of enucleated oocytes receiving cumulus nuclei developed in vitro. Once transferred, many of these embryos implanted and, although most were subsequently resorbed, a significant proportion (2 to 2.8%) developed to term. These experiments show that for mammals, nuclei from terminally differentiated, adult somatic cells of known phenotype introduced into enucleated oocytes are capable of supporting full development.
Comments: Comment in: Nature 1998 Jul 23;394(6691):303
PMID: 9690471, UI: 98352781Comment: The "Honolulu" technique of direct nuclear transfer appears to have been integral to the success of this experiment. At the time, the common view was that mice would be next to impossible to clone, since mouse zygotes show very early activation of the embryonic genome (2-4 cell stage), giving less time for "nuclear re-programming" to occur. This study also demonstrates the higher developmental potential of cumulus cells.
Nat Genet 1999 Jun;22(2):127-8
Cloning of male mice from adult tail-tip cells.
Wakayama T, Yanagimachi RNo abstract available.
PMID: 10369248, UI: 99295922NOTES: First report of cloned MALE animals.
COMMENT: Pending
CLONING Volume 1, Number 3, 1999 Mary Ann Liebert, Inc.
Mammalian Leukocytes Contain All the Genetic Information Necessary for the Development of a New Individual
CESARE GALLI,1 ROBERTO DUCHI,1 ROBERT M. MOOR,2 and GIOVANNA LAZZARI 1We have used leukocytes and oocytes from commercially slaughtered animals to clone a progeny tested Brown Swiss bull. Mononuclear cells were separated from the heparinized blood of the donor male on a Histopaque gradient and cryopreserved. The nuclei of thawed leukocytes were directly microinjected into enucleated Holstein Friesian oocytes that were subsequently activated. Development to morula was 23% and to blastocysts was 17%. Some of the cloned compacting morulae were subjected to a second round of nucleus transfer by fusion of individual blastomeres to enucleated oocytes. Development of these second generation embryos to the blastocyst stage was 19%. Following embryo transfer of 50 blastocysts to 50 recipient heifers (31 from first generation and 19 from second generation), 28 pregnancies were established as evidenced by fetal heartbeat at 35 days. A high proportion of the pregnancies established were lost by day 45. One fetus from a second generation embryo developed to
term. The phenotype (Brown Swiss) and DNA analysis (11 microsatellites on 11 different chromosomes) of the resultant normal healthy calf confirmed its identity to the donor sire. The ability to clone animals from hematopoietic cells that can be easily collected and cryopreserved from any donor irrespective of species, age, or sex has important implications for the preservation of genetic resources from a wide variety of animals in the animal breeding and artificial insemination industries and for human medicine.Quotes & Notes:"... a high probability exists that the nuclei were in a G0 to early G1 phase at transplantation, as virtually all circulating mononuclear cells are normally in a resting or inactive state with levels of proliferating cell nuclear antigen (PCNA) and cyclin indicative of G0 cells (Mathews et al., 1984; Huang et al., 1999)."
Comment:
The cells they used were frozen and then thawed: a process known to negatively impact cellular physiology. It is not unreasonable to conclude that their results would have been better using fresh non-frozen/thawed cells.Direct nuclear transfer was used rather than fusion, since the "leukocytes" were small (6-8 microns)
The second stage nuclear transfer, when done, was performed using electrofusion.From the description, they appear to have selected lymphoctes. Various types of lymphocytes circulate in the peripheral blood: T-cells, B-cells, NK-cells to name a few. This morphological group would also include the very rare hematopoetic pregenitor and stem cells (cd34+), which may very well represent a superior donor cell population.
Science 1998 Dec 11;282(5396):2095-8
Eight calves cloned from somatic cells of a single adult.
Kato Y, Tani T, Sotomaru Y, Kurokawa K, Kato J, Doguchi H, Yasue H, Tsunoda Y
Eight calves were derived from differentiated cells of a single adult cow, five from cumulus cells and three from oviductal cells out of 10 embryos transferred to surrogate cows (80 percent success). All calves were visibly normal, but four died at or soon after birth from environmental causes, and postmortem analysis revealed no abnormality. These results show that bovine cumulus and oviductal epithelial cells of the adult have the genetic content to direct the development of newborn calves.
Comment in: Science 1998 Dec 11;282(5396):1975-6
PMID: 9851933, UI: 99069630Quotes & Notes:Different cell types were used, cumulus cells proving to have significantly better results than the other cell type tested.The authors speculated that Telomerase expression may play a role in the suitability of cells for SCNT. ( In some species cumulus cells are known to express telomerase).
The authors never published the details of their culture system for oocytes.
Comment: Please note the rather AMAZING success rate of 80%. This is the highest of any published report by a wide margin. Also , these calves were given absolutely NO supportive care after birth. There were no pathological abnormalities found in the dead calves. This study re-confirms the efficacy of using cumulus cells for SCNT.
J Reprod Fertil 2000 Nov;120(2):231-237
Cloning of calves from various somatic cell types of male and female adult, newborn and fetal cows.
Kato Y, Tani T, Tsunoda Y
Laboratory of Animal Reproduction, College of Agriculture, Kinki University, 3327-204, Nakamachi,Nara, 631-8505, Japan.
Twenty-four calves were cloned from six somatic cell types of female and male adult, newborn and fetal cows. The clones were derived from female cumulus (n = 3), oviduct (n = 2) and uterine (n = 2) cells, female and male skin cells (n = 10), and male ear (n = 5) and liver (n = 2) cells. On the basis of the number of cloned embryos transferred (n = 172) to surrogate cows, the overall rate of success was 14%, but based on the number of surrogate mothers that became pregnant (n = 50), the success rate was 48%. Cell nuclei from uterus, ear and liver cells, which have not been tested previously, developed into newborn calves after nuclear transfer into enucleated oocytes. To date, seven female and six male calves have survived: six of the females were from adult cells (cumulus (n = 3), oviduct (n = 2) and skin (n = 1) cells) and one was from newborn skin cells, whereas the male calves were derived from adult ear cells (n = 3), newborn liver and skin cells (n = 2), and fetal cells (n = 1). Clones derived from adult cells frequently aborted in the later stages of pregnancy and calves developing to term showed a higher number of abnormalities than did those derived from newborn or fetal cells. The telomeric DNA lengths in the ear cells of three male calves cloned from the ear cells of a bull aged 10 years were similar to those of the original bull. However, the telomeric DNA lengths from the white blood cells of the clones, although similar to those in an age-matched control, were shorter than those of the original bull, which indicates that telomeric shortening varies among tissues.
PMID: 11058438
Quotes & Notes:Review abnormalities
Comment: Mature lymphocytes circulating in the peripheral blood are the product of massive "clonal" expansion, as part of the bodies process of generating immune cells capable of dealing with virtually any foreign antigen. So it is not surprising to see "shortened" telomeres in such cells, even though lymphocytes are known to express telomerase to varying extents. Hematopoeitic stem cells, from which ALL circulating leukocytes are derived, are known to express relatively high levels of telomerase. They are also known to already exist largely in the quiescent G0/G1 phase in the peripheral circulation.
Science 1998 May 22;280(5367):1256-8
Cloned transgenic calves produced from nonquiescent fetal fibroblasts.
Cibelli JB, Stice SL, Golueke PJ, Kane JJ, Jerry J, Blackwell C, Ponce de Leon FA, Robl JMDepartment of Veterinary and Animal Sciences, University of Massachusetts, Amherst, MA 01003, USA.
An efficient system for genetic modification and large-scale cloning of cattle is of importance for agriculture, biotechnology, and human medicine. Here, actively dividing fetal fibroblasts were genetically modified with a marker gene, a clonal line was selected, and the cells were fused to enucleated mature oocytes. Out of 28 embryos transferred to 11 recipient cows, three healthy, identical, transgenic calves were generated. Furthermore, the life-span of near senescent fibroblasts could be extended by nuclear transfer, as indicated by population doublings in fibroblast lines derived from a 40-day-old fetal clone. With the ability to extend the life-span of these primary cultured cells, this system would be useful for inducing complex genetic modifications in cattle.
Comment in: Science 1998 Sep 11;281(5383):1611
PMID: 9596577, UI: 98259082Quotes & Notes:Pending
Comment: Particular conditions of cell culture appear to cause some degree of "nuclear re-programming" to occur in culture (see next reference also), rendering the nuclear material more capable of directing proper embryonic development following SCNT.Could this be due to induction of telomerase activity?
And perhaps synthesis of new, relatively acetylated forms of histone protein?
Can the acetylation state of newly formed histones be influenced by culture conditions?
Perhaps by adding acetyl-group donors? Such as pantothenic acid [ 1, 2 ]?
Culture conditions must also be idealized to minimize oxidative and photonic damage.
Proc Natl Acad Sci U S A 2000 Feb 1;97(3):990-5 LINK TO FULL TEXT ARTICLE
Six cloned calves produced from adult fibroblast cells after long-term culture.
Kubota C, Yamakuchi H, Todoroki J, Mizoshita K, Tabara N, Barber M, Yang X
Cloning whole animals with somatic cells as parents offers the possibility of targeted genetic manipulations in vitro such as "gene knock-out" by homologous recombination. However, such manipulation requires prolonged culture of nuclear donor cells. Previous successes in cloning have been limited to the use of cells collected either fresh or after short-term culture. Therefore, demonstration of genetic totipotency of cells after prolonged culture is pivotal to combining site-specific genetic manipulations and cloning. Here we report birth of six clones of an aged (17-year-old) Japanese Black Beef bull using ear skin fibroblast cells as nuclear donor cells after up to 3 months of in vitro culture (10-15 passages). We observed higher developmental rates for embryos derived from later passages (10 and 15) as compared with those embryos from an early passage (passage 5). The four surviving clones are now 10-12 months of age and appear normal, similar to their naturally reproduced peers. These data show that fibroblasts of aged animals remain competent for cloning, and prolonged culture does not affect the cloning competence of adult somatic donor cells.
Comments: Proc Natl Acad Sci U S A 2000 Feb 1;97(3):956-7
PMID: 10655472, UI: 20122559Quotes & Notes:Pending
Comment: See preceding reference comment.
Reprod Fertil Dev 1998;10(4):369-78
Adult somatic cell nuclear transfer is used to preserve the last surviving cow of the Enderby Island cattle breed.
Wells DN, Misica PM, Tervit HR, Vivanco WHReproductive Technologies Group, AgResearch, Ruakura Research Centre, Hamilton,
New Zealand. wellsd@agresearch.cri.nzTo preserve the female genetics of an endangered breed of cattle, adapted to sub-Antarctic conditions, adult somatic cell nuclear transfer was used to clone the last surviving Enderby Island cow from mural granulosa cells. Embryos reconstructed with metaphase II cytoplasts and quiescent cells were either activated and fused simultaneously (AFS) at 24 or 30 hours post maturation (hpm) or alternatively, fused 4-6 h before activation at 26-30 hpm (FBA). A significantly higher proportion of fused embryos developed in vitro to grade 1-3 blastocysts on Day 7 with FBA (39.8+/-2.8%) compared to AFS with activation either at 24 hpm (10.6+/-3.9%, P<0.01) or at 30 hpm (18.6+/-4.1%, P<0.01). Following the transfer of 74 embryos from the FBA treatment over two experiments, survival rates on Days 30, 55, 85, 150 and 190 of pregnancy were 38%, 30%, 23%, 16% and 15%, respectively. Of 22 embryos transferred in the first experiment, two calves were born alive with one calf surviving. DNA analyses confirmed that the calves were genetically identical to the Enderby Island cow. Additional pregnancies are currently ongoing. These data show that embryo development is increased by prolonged exposure of quiescent somatic cell nuclei to oocyte cytoplasm before artificial activation, possibly facilitating nuclear reprogramming. The successful demonstration of somatic cell nuclear transfer in animal conservation extends the applications of the technology beyond the main agricultural and biomedical interests.
PMID: 10355689, UI: 99281820Quotes & Notes:Pending
Comment: A time delay between fusion or insertion of the nuclear donor material appears to facilitate nuclear reprogramming. Other studies have also demonstrated this effect.
Biol Reprod 1999 Apr;60(4):996-1005
Production of cloned calves following nuclear transfer with cultured adult mural granulosa cells.
Wells DN, Misica PM, Tervit HR.AgResearch, Ruakura Research Centre, PB 3123, Hamilton, New Zealand. wellsd@agresearch.cri.nz
Adult somatic cell nuclear transfer was used to determine the totipotent potential of cultured mural granulosa cells, obtained from a Friesian dairy cow of high genetic merit. Nuclei were exposed to oocyte cytoplasm for prolonged periods by electrically fusing quiescent cultured cells to enucleated metaphase II cytoplasts 4-6 h before activation (fusion before activation [FBA] treatment). Additionally, some first-generation morulae were recloned by fusing blastomeres to S-phase cytoplasts. A significantly higher proportion of fused embryos developed in vitro to grade 1-2 blastocysts on Day 7 with FBA (27.5 +/- 2.5%) than with recloning (13.0 +/- 3.6%; p < 0. 05). After the transfer of 100 blastocysts from the FBA treatment, survival rates on Days 60, 100, 180, and term were 45%, 21%, 17%, and 10%, respectively. Ten heifer calves were delivered by elective cesarean section; all have survived. After the transfer of 16 recloned blastocysts, embryo survival on Day 60 was 38%; however, no fetuses survived to Day 100. DNA analyses confirmed that the calves are all genetically identical to the donor cow. It is suggested that the losses throughout gestation may in part be due to placental dysfunction at specific stages. The next advance in this technology will be to introduce specific genetic modifications of biomedical or agricultural interest.
PMID: 10084977
J Reprod Fertil 1999 Mar;115(2):325-31
Effects of serum starvation and re-cloning on the efficiency of nuclear transfer using bovine fetal fibroblasts.
Zakhartchenko V, Durcova-Hills G, Stojkovic M, Schernthaner W, Prelle K, Steinborn R, Muller M, Brem G, Wolf E.
Bavarian Research Centre for Biology of Reproduction (BFZF), Ludwig-Maximilian University, Oberschleissheim, Germany.The developmental potential of bovine fetal fibroblasts was evaluated using nuclear transfer. Fibroblasts from a 37-day-old fetus were fused to enucleated oocytes before activation. Nuclei of starved (cultured for 8 days in medium containing 0.5% serum) fibroblasts supported the development of reconstructed embryos to the blastocyst stage significantly better than those of non-starved fibroblasts (39% versus 20%; P < 0.05). When nuclear transfer morulae derived from starved or non-starved fibroblasts were used for re-cloning, the proportion of blastocysts (52 and 55%, respectively) obtained with these embryonic nuclei was significantly higher than it was with fibroblast nuclei used in the first round of nuclear transfer (P < 0.05 and P < 0.001, respectively). After transfer of blastocysts derived from non-starved and starved fibroblasts, respectively, 33% (1/3) and 78% (7/9) of recipients were pregnant on day 30 as assessed by ultrasonography. On day 90, the corresponding pregnancy rates were 33% (1/3) and 63% (5/8). Two live male twin calves, derived from non-starved fibroblasts, were delivered by Caesarean section at day 281 of gestation. This study demonstrates a positive effect of serum starvation on the efficiency of nuclear transfer using bovine fetal fibroblasts. The efficiency of nuclear transfer could be further increased by recloning.
PMID: 10434938
Comment: second stage nuclear transfer ( recloning) significantly increases efficiency and developmental potential.
Nature 2000 Sep 7;407(6800):86-90
Cloned pigs produced by nuclear transfer from adult somatic cells.
Polejaeva IA, Chen SH, Vaught TD, Page RL, Mullins J, Ball S, Dai Y, Boone J, Walker S, Ayares DL, Colman A, Campbell KH
PPL Therapeutics Incorporated, Blacksburg, Virginia 24060, USA. ipolejaeva@ppl-therapeutics.comSince the first report of live mammals produced by nuclear transfer from a cultured differentiated cell population in 1995 (ref. 1), successful development has been obtained in sheep, cattle, mice and goats using a variety of somatic cell types as nuclear donors. The methodology used for embryo reconstruction in each of these species is essentially similar: diploid donor nuclei have been transplanted into enucleated MII oocytes that are activated on, or after transfer. In sheep and goat pre-activated oocytes have also proved successful as cytoplast recipients. The reconstructed embryos are then cultured and selected embryos transferred to surrogate recipients for development to term. In pigs, nuclear transfer has been significantly less successful; a single piglet was reported after transfer of a blastomere nucleus from a four-cell embryo to an enucleated oocyte; however, no live offspring were obtained in studies using somatic cells such as diploid or mitotic fetal fibroblasts as nuclear donors. The development of embryos reconstructed by nuclear transfer is dependent upon a range of factors. Here we investigate some of these factors and report the successful production of cloned piglets from a cultured adult somatic cell population using a new nuclear transfer procedure.
Comment in: Nature 2000 Sep 7;407(6800):27, 29-30
PMID: 10993078, UI: 20445174Quotes & Notes: Factors contributing to success:
1. Employed contact inhibition as a means of generating quiescent cells for fusion.Comment:
2. Time delay between fusion and activation of constructs.
3. Utilized a new second stage nuclear transfer method ( re-cloning)This experiment is particularly significant in that multiple cohorts utilizing different variables were studied. The ONLY cohort to produce live ( AND PERFECTLY HEALTHY) offspring was the combination of the above 3 mentioned factors.
Bringing cultured cells to quiescence is better achieved through growth to contact inhibition, rather than through serum starvation as initially employed by Wilmut. Serum starvation appears to result in more DNA damage.
Cloning: Volume: 1 Number: 1 Page: 63 -- 69 RETRIEVE FREE FULL TEXT ARTICLE
Reprogramming of Fibroblast Nuclei after Transfer into Bovine Oocytes
Paul A. De Sousa; Quinton Winger; Jonathan R. Hill; Karen Jones; Andrew J. Watson; Mark E. Westhusin
DOI: 10.1089/15204559950020102 Publisher: Mary Ann Liebert, Inc.
Recent landmark achievements in animal cloning have demonstrated that the events of cell differentiation can, in principle, be reversed. This reversal necessarily requires large-scale genetic reprogramming, of which little is known. In the present study we characterized the extent to which blastocyst stage-specific mRNA expression would be conserved in bovine embryos produced by nuclear transfer (NT) using fetal fibroblasts as nuclei donors (FF NT). The mRNA pool of FF NT embryos was compared with that of NT embryos reconstructed from embryonic blastomeres (Emb NT), with embryos produced under in vivo or in vitro conditions, and finally with fibroblast cells. Embryo/cell-specific mRNA pools were contrasted using differential display methodology. Random oligonucleotide primer pair combinations were used to subfractionate mRNA populations and represent individual mRNAs as copy DNA (cDNA) bands ranging in size from 100 to 800 base pairs. Regardless of whether bovine blastocysts developed in vivo or in vitro, or were derived after nuclear transplantation with embryonic blastomeres or fetal fibroblasts, their mRNA profile was highly conserved and distinct from that of fetal fibroblast cells. There was approximately 95% conservation in cDNA banding patterns between FF NT, Emb NT, and in vivo derived blastocysts, when compared with in vitro derived blastocysts. In contrast, the cDNA banding in fibroblasts was only 67% conserved with in vitro derived blastocysts (p < 0.0001), indicating that dramatic changes in gene transcription are induced by nuclear transplantation. After nuclear transplantation, gene expression in fetal fibroblasts is reprogrammed so to mimic that of preimplantation embryo development. Future characterization of these changes will be invaluable for the identification of suitable cell types to serve as nuclear donors for embryo reconstruction and provide information that can be used to improve the efficiency of cloning animals by nuclear transplantation.Quotes & Notes:Pending
Comment: Is there is a quantitative as well as a qualitative aspect to successful nuclear reprogramming. Almost any donor cell type may be suitable if grown in an appropriate cell culture system which minimizes DNA damage and "promotes" nuclear reprogramming.
Proc Natl Acad Sci U S A 1999 May 25;96(11):5894-6 VIEW FULL TEXT ARTICLE
DNA demethylation.
Wolffe AP, Jones PL, Wade PALaboratory of Molecular Embryology, National Institute of Child Health and Human Development, National
Institutes of Health, Bethesda, MD 20892-5431, USA.".... DNA methylation is remarkably dynamic during early mammalian development and in certain tumor cells (12, 13). Alterations in the methylation status of the entire genome (14, 15), individual chromosomes (16), and specific genes (17-20) are essential for normal development (21, 22) and can promote tumorigenesis (23, 24)."
"With respect to more global genome demethylation, particular histone modifications such as acetylation might exert a "teflon effect," preventing efficient access of the methyltransferase enzyme. These models predict that DNA replication will be an integral component of demethylation."
"DNA methylation dynamics present a mystery that needs to be solved."
Publication Types: Comment Review Review, tutorial
Comment on:
Proc Natl Acad Sci U S A. 1999 May 25;96(11):6107-12
PMID: 10339513
MCB -- Araujo et al. 18 (6): 3475"Although methylated cytosines, in contrast to other forms of epigenetic control, are part of the covalent structure of the genome, they are inherited by a postreplicative enzymatic transfer of methyl groups from S-adenosyl methionine, which is catalyzed by DNA methyltransferase (MeTase) (1)"
Biochim Biophys Acta 1979 Feb 27;561(2):345-57
Mouse DNA methylase: methylation of native DNA.
Adams RL, McKay EL, Craig LM, Burdon RHNotes:
"The preferred double stranded DNA substrate is that rendered partially unmethylated by an in vitro repair mechanism or by isolation from methionine starved cells. "Comment:
DNA methylation can be influenced by culture conditions.
Induction of specific DNA repair processess can induce a partially unmethylated state of DNA (local?vs global)
Can one conclude that oxidative damage stimulates DNA repair mechanisms, and without subsequent remethylation of those sites results in abberant gene expression?
Science 2000 Sep 29;289(5488):2360-2
Active remodeling of somatic nuclei in egg cytoplasm by the nucleosomal ATPase ISWI.
Kikyo N, Wade PA, Guschin D, Ge H, Wolffe APLaboratory of Molecular Embryology, Building 18T, Room 106, National Institutes of Health, Bethesda, MD 20892, USA.
Cloning by the transplantation of somatic nuclei into unfertilized eggs requires a dramatic remodeling of chromosomal architecture. Many proteins are specifically lost from nuclei, and others are taken up from the egg cytoplasm. Recreating this exchange in vitro, we identified the chromatin-remodeling nucleosomal adenosine triphosphatase (ATPase) ISWI as a key molecule in this process. ISWI actively erases the TATA binding protein from association with the nuclear matrix. Defining the biochemistry of global nuclear remodeling may facilitate the efficiency of cloning and other dedifferentiation events that establish new stem cell lineages.
PMID: 11009424Quotes & Notes:"During the remodeling process, somatic cell nuclei transplanted into Xenopus eggs lose >85% of radiolabeled protein concomitant with substantial uptake of proteins from the egg cytoplasm(4). This protein exchange is likely to be mechanistically involved in nuclear remodeling."Comment: Nuclear reprogramming in an energy dependant process requiring ATP."..remodeling of local chromatin and DNA methylation states requires the activity of nucleosomal ATPases (16-20,32)"
Crit Rev Eukaryot Gene Expr 2000;10(1):1-12
Understanding "active" chromatin: a historical perspective of chromatin remodeling.
Krebs JE, Peterson CLDepartment of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester 01605, USA.
Two phenomena have long been observed to correlate with transcriptionally active chromatin: increased histone acetylation and increased sensitivity to nucleases, including specific patterns of nuclease hypersensitivity in the promoters of active or inducible genes. Work in recent years has at last identified protein complexes required to form these hallmarks of active chromatin: histone acetyltransferases (HATs) and ATP-dependent chromatin remodeling complexes. This review traces the history of these discoveries, including the development of essential tools that allowed the major advances in the field, and describes the current understanding of the interactions between HATs and ATP-dependent remodelers.
PMID: 10813389Quotes & Notes: Pending
Comment: Increased histone acetylation is correlated with transcriptionally active chromatin.
Chem Biol 1997 Dec;4(12):885-8
Protein acetylation: more than chromatin modification to regulate transcription.
Bayle JH, Crabtree GRDepartment of Pathology, Beckman Center for Molecular and Genetic Medicine,
Stanford University School of Medicine, Palo Alto, CA 94305, USA.Histone acetyltransferases and deacetylases are involved in the regulation of gene transcription. Recently, tumor suppressor protein p53 has been shown to be a target for transcriptional coactivators that have histone acetyltransferase activity, suggesting acetylation is also involved in the regulation of cell proliferation and tumorigenesis.
PMID: 9427654Quotes & Notes:Pending
Comment: "suggesting acetylation is also involved in the regulation of cell proliferation" - something immensely crucial to embryonic development.Is this the reason that cultured cells regain an ability to direct embryoninc development? New histone proteins must be made for cells to grow and divide. Thus the newly generated histone complexs may show a higher level of acetylation, and can this be influenced or promoted by appropriate culture conditions? ( see pantothenic acid)
Cell Mol Life Sci 1998 Jan;54(1):6-20
Linking histone acetylation to transcriptional regulation.
Mizzen CA, Allis CDDepartment of Biology, University of Rochester, New York 14627, USA.
In eukaryotes, DNA is assembled with histones to form nucleosomes, the basic subunit of chromatin structure. The wrapping of DNA around histone octamers to form nucleosomal filaments and further folding of these filaments are necessary to contain eukaryotic genomes within nuclei. However, the dense packing of chromatin in nuclei and the association of DNA with histones restrict the access of proteins involved in gene transcription to DNA. Abundant biochemical data supports a long-standing correlation between histone acetylation and gene activation, suggesting that histone acetylation acts to enhance the access of transcription-associated proteins to DNA. However, despite this correlation, nuclear enzymes responsible for transcription-associated histone acetylation have been identified only recently. Here we review evidence suggesting that histone acetylation represents a major pathway for transcriptional regulation, and discuss possible roles for transcription-associated histone acetyltransferases in this regulation.
Review, tutorial
PMID: 9487383Quotes & Notes:Pending
Comment: The acetylated form of histone is associated with enhanced gene transciption.
Genes Dev 1999 Sep 15;13(18):2339-52 LINK TO FULL TEXT ARTICLE
ATP-dependent remodeling and acetylation as regulators of chromatin fluidity.
Kingston RE, Narlikar GJDepartment of Molecular Biology, Massachusetts General Hospital, Boston,
Massachusetts, 02114 USA; Department of Genetics, Harvard Medical School, Boston,
Massachusetts 02115 USA. kingston@frodo.mgh.harvard.edu
Review, academic
PMID: 10500090Article Conclusions: " It is apparent that chromatin structure is dynamic and that structural changes in chromatin are highly regulated. Current data suggest that one role of ATP-dependent remodeling complexes is to use the energy of ATP hydrolysis to increase the rate at which different structures interchange. These complexes thus make chromatin more fluid. Mechanisms must also exist to fix genes in an active or a repressed state; here, acetylation and deacetylation complexes might be key players. Thus, both the rate at which a chromatin remodeling event can occur and the thermodynamic stabilities of the end products are essential to regulation. It appears as if nature has devised large families of complexes to govern both aspects of chromatin dynamics, allowing an exquisite regulation of chromatin structures. "Comment: This paper presents a very elegant model of "chromatin fluidity", once again an energy driven process utilizing ATP. Optimization of intracellular ATP levels should theoretically facilitate nuclear reprogramming. Adding a special form of CoQ10 to the culture media has been shown to increase intracellular ATP levels and to promote better development of non-SCNT blastocysts. A quantitative aspect is also implied.
Semin Perinatol 2000 Feb;24(1):46-50
The role of nitric oxide in apoptosis.
Li J, Billiar TRDepartment of Surgery, University of Pittsburgh School of Medicine, PA, USA.
Nitric oxide (NO) has been shown to play important roles in modulating cell viability. Both proapoptotic and antiapoptotic functions of NO have been reported. This article discusses our current understanding on the mechanisms responsible for the dual actions of NO in regulating apoptosis and propose that one common mechanism for NO-mediated inhibition of apoptosis is the inhibition of caspases via nitrosylation.
PMID: 10709859, UI: 20173093
Comment: Apoptosis is known to occur in developing embryos, both in IVF and SCNT. Would apoptosis be reduced in a developing embryo if activation of the oocyte is through exposure to Nitric Oxide?
Biol Reprod 1999 Jun;60(6):1483-7
Retinol administration to superovulated ewes improves in vitro embryonic viability.
Eberhardt DM, Will WA, Godkin JDDepartment of Animal Science, The University of Tennessee, Knoxville, Tennessee
37901, USA.Retinol and its metabolites, all-trans retinoic acid and 9-cis retinoid acid, are regulators of cellular growth, differentiation, and development and have been implicated in reproductive processes including folliculogenesis and embryonic survival. Three experiments were conducted to identify effects of retinoid treatment of superovulated ewes upon subsequent in vitro embryonic development. Ewes were treated with all-trans retinol (ROH), all-trans retinoic acid (RA), 9-cis retinoic acid (CIS), or vehicle (Control) on the first and last day of FSH treatment. Embryos were recovered at the morula stage, cultured in vitro for 96 h, and observed for blastocyst formation. Embryos from ROH-treated animals had a higher (p < 0.01) incidence of blastocyst formation than RA-, CIS-, or vehicle-treated animals (72% vs. 27%, 33% and 32%, respectively). In experiment 2, ewes were given ROH or vehicle and treated as above. ROH treatment resulted in an increased percentage of embryos forming blastocysts (70% vs. 22%, p < 0.05). In experiment 3, ewes were treated with ROH or vehicle, and embryos were collected at the 1- to 4-cell stage and cultured for 7 days. ROH treatment resulted in increased blastocyst formation (79% vs. 5%, p < 0.05). The majority of embryos (60% vs. 6%; p < 0.01)) from vehicle-treated animals failed to develop beyond the 8-cell stage in comparison with those from ROH animals[1]. ROH treatment of superovulated ewes increased embryonic viability and positively impacted embryonic development.
PMID: 10330109Quotes & Notes:PendingComment: Retinoic acid and it's derivatives are known teratogens. Perhaps the particular form all-trans retinol (ROH) is not when given in the fashion specified above. This study should be repeated with other species, with the resulting blastocysts actually transferred in order to assess development and any possible teratogenic effects. It would also be of interest to assess aneuploidy in this blastocyst population.
[1]Many human IVF embryos similarly arrest at the 8-cell stage...
Fertil Steril 2001 Feb;75(2):348-353 View Full text Article Online ( Subscibers only)
The spindle observation and its relationship with fertilization after intracytoplasmic sperm injection in living human oocytes.
Wang W, Meng L, Hackett RJ, Odenbourg R, Keefe DLDivision of Reproductive Medicine and Infertility, Department of Obstetrics and Gynecology, Women and Infants Hospital of Rhode Island, Brown University School of Medicine, Providence, Rhode Island, USA
Objective: To image spindles in living human oocytes and to examine the relation between spindles and fertilization after ICSI.
Design: The LC polscope was used to examine spindles in an observational study of living oocytes.
Setting: Academic IVF clinic.Patient(s): Women being treated for infertility.
Intervention(s): Oocytes retrieved from patients for infertility treatment were examined before ICSI. Aged, unfertilized oocytes after IVF or ICSI were examined with polscope and confocal microscopes to compare the two methods.
Main Outcome Measure(s): Spindle structure in living oocytes and fertilization after ICSI.
Result(s): Spindles could be imaged in 61.4% of oocytes. More oocytes with spindles than oocytes without spindles fertilized normally after ICSI (61.8% vs. 44.2%). Spindles in most aged oocytes were partially or completely disassembled, and only a few microtubules around the chromosomes or dispersed microtubules in the cytoplasm were observed. Confocal images of immunostained spindles were almost identical to polscope images of spindle birefringence.
Conclusion(s): Spindles in living human oocytes can be imaged by using the polscope. A birefringent spindle in human oocytes may clinically predict the quality and age of oocytes. This method also can be used to monitor spindle position during ICSI.
PMID: 11172838Quotes & Notes:
"An increased incidence of chromosomal aneuploidies is thought to contribute to decreased clinical pregnancy and implantation rates in older women (9, 10). In IVF clinics, such abnormalities have been detected in oocytes from young women as well, especially those with pathologic ovarian conditions (12)""Because the cytoskeleton is sensitive to environmental changes, disruption of spindle architecture may also occur during oocyte maturation under the nonphysiologic conditions presented by the IVF laboratory"
"Battaglia et al. (13) recently reported that the proportion of oocytes with abnormal spindles was significantly higher in older than in young women, and abnormal spindles were associated with abnormal chromosomal distribution."
"The rate of abnormal spindle structure also increases as oocytes age in culture (2, 14, 15)"
"It seems that human oocytes are more sensitive to temperature fluctuations than are oocytes from other animals (3, 6, 8, 22), and spindle structure is disrupted even when oocytes are exposed briefly to temperature even slightly less than 37°C (4).
Comment: Donor oocytes should come from younger individuals without pathologic ovarian conditions. During SCNT, can the nuclear material be removed without removing the spindle? This might address some observations and concerns made by Tanja Dominko regarding SCNT in primates in this article ( no link yet)
Biol Reprod 1997 Jul;57(1):49-53
Synchronization of meiosis in porcine oocytes by exposure to dibutyryl cyclic adenosine monophosphate improves developmental competence following in vitro fertilization.
Funahashi H, Cantley TC, Day BN
Department of Animal Sciences, University of Missouri-Columbia, 65211, USA.The effect of stage of maturation of the germinal vesicle of porcine oocytes at the time of in vitro maturation on subsequent developmental competence was examined. A large variation exists in the germinal vesicle morphology of oocytes at the time of collection of cumulus-oocyte complexes (COCs) and after culture in the absence of dibutyryl cAMP (dbcAMP) for 20 h. However, the morphology of the germinal vesicle was synchronized to a specific stage after culture in the presence of 1 mM dbcAMP for 20 h. There was no difference in germinal vesicle breakdown rate (total mean, 75.0 +/- 5.4%) or in maturation rate (total mean, 82.1 +/- 2.1 %) at 28 and 44 h of culture, respectively. However, differences in meiotic progress of oocytes were observed (p < 0.05) at 36 h of culture when COCs were exposed to dbcAMP for the first 20 h of maturation, as compared to controls. The incidence of embryos that developed to the blastocyst stage after in vitro fertilization was higher (p < 0.05) when COCs were exposed to dbcAMP (21.5 +/- 2.5%) as compared to controls (9.2 +/- 1.6%). After transfer of experimental embryos to four recipient gilts, the three pregnant recipients delivered 19 live piglets. These results indicate that exposure of COCs to dbcAMP for the first 20 h of culture for maturation increases the homogeneity of oocyte nuclear maturation and improves the efficiency of in vitro production of swine embryos.
PMID: 9209079, UI: 97352720Quotes & Notes:Pending
Comment: Is there rational for repeating this study with other species?
Hum Reprod 2000 May;15(5):1149-54
Chemically and mechanically induced membrane fusion: non-activating methods for nuclear transfer in mature human oocytes.
Tesarik J, Nagy ZP, Mendoza C, Greco ELaboratoire d'Eylau, 55 rue Saint-Didier, 75116 Paris, France.
Most current studies of nuclear transfer in mammalian oocytes have used electrofusion to incorporate donor cell nuclei into enucleated oocyte cytoplasts. However, the application of electrofusion to human oocytes is hampered by the relative ease with which this procedure induces oocyte activation. Here we tested a previously described chemical fusion technique and an original mechanical fusion procedure in this application. Enucleated metaphase II oocytes were first agglutinated with karyoplasts originating from other metaphase II oocytes and then induced to fuse with the use of polyethylene glycol or by micromanipulation with an intracytoplasmic sperm injection (ICSI) micropipette. Both techniques yielded a high frequency of fusion and did not cause oocyte activation. Moreover, the reconstructed oocytes were easily activated by subsequent treatment with ionophore A23187 and 6-dimethylaminopurine. These techniques may be used in attempts to alleviate female infertility due to insufficiency of ooplasmic factors by nuclear transfer from patients' oocytes to enucleated donor oocyte cytoplasts. For eventual future use in human cloning, they would ensure prolonged exposure of transferred nuclei to metaphase promoting factor, which appears to be required for optimal nuclear reprogramming.
PMID: 10783368, UI: 20247308
Quotes & Notes:Pending
Comment: This author suggests a quantitative aspect to successful nuclear reprogramming. Is this a possible explanation for the observed higher rates of embryonic development seen in "recloned" embryos ( 2nd stage nuclear transfer ) using various strategies (Link1)? J.Cohen has also pursued ooplasmic transfer as a method to increase the developmental viability of oocytes, though not without the controversy of generating mitochondrial "heteroplasmy" as a consequence. That could be avoided by simply transferring ooplasm between oocytes of the same donor.
General
Res Exp Med (Berl) 1988;188(5):391-6
Effects of pantothenic acid on fibroblastic cell cultures.
Lacroix B, Didier E, Grenier JFInserm Unite 61, Hopital Civil, Strasbourg, France.
To evaluate the effects of pantothenic acid during wound healing processes, fibroblastic cell cultures originating from foreskin were established and subcultured by trypsinization. PA (40 micrograms/ml) was added to the basal
culture medium. The cell proliferation was estimated by cell count and determination of 3H-thymidine incorporation. The protein synthesis and secretion were determined by dosage in the cells and in the culture medium. When PA was added to the medium, a significant increase of cell proliferation and of 3H-thymidine incorporation was observed mainly during the first few days. PA also stimulated intracellular protein synthesis, but did not induce a release of proteins in the culture medium. The exact mechanism involved in this phenomenon remains unclear at this time.
PMID: 3227160Quotes & Notes:Pending
Comment: One of the best criteria to assess blastocyst quality is cell number
Hematopoeitic Stem and Progenitor Cells
Exp Hematol 1998 Feb;26(2):143-157
Identification of distinct elements of the stromal microenvironment that control human hematopoietic stem/progenitor cell growth and differentiation.
Aiuti A, Friedrich C, Sieff CA, Gutierrez-Ramos JC
Center for Blood Research, Inc., Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.Using a novel collection of conditionally immortalized mouse stromal cell clones, we evaluated the role of distinct elements of the hematopoietic microenvironment in supporting and regulating the growth, division, and differentiation of a candidate human stem cell population (CD34+/CD38-). We found functional diversity in the capacity of different stromal cell clones to support the growth of primitive (CD34+/CD38-) and committed (CD34+/CD38+) hematopoietic progenitors and their differentiation into mature hematopoietic cells (CD34-/CD45+). Among the stromal cell clones that supported long-term hematopoiesis, we identified two clones that induced expansion of CD34+ progenitor/stem cells during the first 4 weeks of coculture and that supported the maintenance of this CD34+ population for up to 10 weeks in vitro. However, these two clones appeared to represent two different microenvironments with regard to the signals they provide to the different CD34+ progenitor subpopulations: One stromal clone preserved a pool of undifferentiated, relatively quiescent (CD34+/CD38-) progenitor cells, allowing their differentiation at a low rate into more committed (CD34+/CD38+) progenitors; the other fostered a more extensive and rapid differentiation of all CD34+/CD38- progenitors into CD34+/CD38+ cells, preferentially maintaining this committed population at a higher rate of cell division. These stromal cell clones were also able to support the proliferation and differentiation of CD34+/CD38- cells in conditions in which progenitor-stroma contact was prevented. This collection of stromal cell clones may represent a unique tool for the study of stromal regulators of hematopoiesis as well as for the support of gene transfer into hematopoietic progenitor cells.
PMID: 9472804, UI: 98132146
Comment: CD34+ hematopoitic progenitor and stem cells may be isolated from the peripheral blood of adults, either through pheresis procedure, in in small numbers by using immunomagnetic beads.
Vox Sang 1998;74 Suppl 2:91-4
Stem cell biology for the transfusionist.
Lansdorp PM
Terry Fox Laboratory, British Colombia Cancer Agency, Vancouver, Canada. peter@terryfox.ubc.caThe limited life-span of most blood cells requires continuous production of cells which in adults may exceed 1012 cells/day. This impressive production of cells (approximately 4.1015 cells over a life time) is achieved by the proliferation and differentiation of committed progenitor cells which themselves are derived from a population of pluripotent stem cells with self-renewal potential. In adults, the large majority of stem cells are found in the bone marrow among cells with a CD34 + CD38- phenotype. Interestingly, small but significant numbers of such cells can be found in the circulation. The frequency of circulating CD34 + CD38- cells can be dramatically increased by treatment with certain compounds including cytokines. Such "mobilized" peripheral blood stem cells have become an important alternative to bone marrow in stem cell transplantation procedures primarily because engraftment is more rapid. The latter is almost certainly related to the increased numbers of primitive CD34 + CD38- cells capable of engrafting the bone marrow in blood versus bone marrow stem cell grafts [1]. Paradoxically, the large majority of "candidate" stem cells in adult bone marrow are quiescent cells.One possibility is that stem cells, like other somatic cells, have only a limited replicative potential (< 100 divisions). This hypothesis is supported by two key observations and the consideration that, in theory, 52 divisions can yield 4.1015 cells. First, it was shown that "candidate" stem cells purified from fetal and adult tissue display marked functional differences in turn-over time and the ability to produce cells with stem cell properties [2]. Secondly, these functional differences were found to correlate with a measurable loss of telomere repeats [3], despite the presence of low but readily detectable levels of telomerase in all purified cell fractions [4,5]. In order to address questions about the role of telomeres in normal and malignant hematopoiesis, we developed quantitative fluorescence in situ hybridization [6]. With this technique the length of telomere repeats at individual chromosome ends can be reliably estimated using optical density measurements from digital images of metaphase chromosomes after fluorescence in situ hybridization with directly labeled (CCCTAA)3--Peptide Nucleic Acid Probe [6,7]. Furthermore, we recently showed that this method can be adapted to measure the total telomere repeat content of cells by flow cytometry [8]. Here some issues in studies of hematopoietic stem cells are discussed in relation to rapidly accumulating information about telomere biology.
PMID: 9704429, UI: 98370052
Blood 1997 Jun 15;89(12):4337-47
In vitro maintenance of highly purified, transplantable hematopoietic stem cells.
Moore KA , Ema H , Lemischka IR
Department of Molecular Biology, Princeton University, NJ 08544, USA.The cellular and molecular mechanisms that regulate the most primitive hematopoietic stem cell are not well understood. We have undertaken a systematic dissection of the complex hematopoietic microenvironment to define some of these mechanisms. An extensive panel of immortalized stromal cell lines from murine fetal liver were established and characterized. Collectively, these cell lines display extensive heterogeneity in their in vitro hematopoietic supportive capacity. In the current studies, we describe a long-term in vitro culture system using a single stromal cell clone (AFT024) that qualitatively and quantitatively supports transplantable stem cell activity present in highly purified populations. We show multilineage reconstitution in mice that received the equivalent of as few as 100 purified bone marrow and fetal liver stem cells cultured for 4 to 7 weeks on AFT024. The cultured stem cells meet all functional criteria currently ascribed to the most primitive stem cell population. The levels of stem cell activity present after 5 weeks of coculture with AFT024 far exceed those present in short-term cytokine-supported cultures. In addition, maintenance of input levels of transplantable stem cell activity is accompanied by expansion of other classes of stem/progenitor cells. This suggests that the stem/progenitor cell population is actively proliferating in culture and that the AFT024 cell line provides a milieu that stimulates progenitor cell proliferation while maintaining in vivo repopulating activity.
Article Number: UI97335961
Comment: Here is a culture system to maintain hematopoitic stem cells.
Exp Hematol 1998 Jul;26(7):612-9
The murine stromal cell line AFT024 acts specifically on human CD34+CD38- progenitors to maintain primitive function and immunophenotype in vitro.
Thiemann FT, Moore KA, Smogorzewska EM, Lemischka IR, Crooks GM
Division of Research Immunology and Bone Marrow Transplantation, Childrens Hospital Los Angeles, California 90027, USA.A stromal cell line derived from murine fetal liver (AFT024) has been demonstrated to maintain long-term repopulating murine stem cells for up to 7 weeks in vitro. We evaluated the ability of AFT024 to maintain the immunophenotype and function of primitive human progenitors in vitro by comparing the cocultivation of CD34+CD38 cells on AFT024 with that on primary human stroma (HS). We have previously reported that within the CD34+CD38- population of bone marrow and cord blood, a highly primitive progenitor subpopulation can be identified functionally by its ability to generate colony forming unit-cells (CFU-Cs) in extended long-term culture (ELTC), that is, beyond 60 days of stromal cocultivation. Cocultivation of bone marrow and cord blood CD34+CD38-cells on AFT024 produced significantly greater cell expansion (p=0.0002) and CFU-C output (p=0.0007) during the ELTC period compared with culturing on HS. CFU-C production continued up to 9 weeks longer on AFT024 stroma. After 3 to 4 weeks of bulk culture on either AFT024 or HS, cells were replated in a limiting dilution to measure the number of cobblestone area-forming cells (CAFCs) maintained on each stroma. AFT024 maintained significantly more CAFCs than did HS (n=3, p=0.002). Fluorescence-activated cell sorter analysis of AFT024 and HS cocultures showed that both the frequency (p=0.018) and absolute number (p=0.027) of CD34+CD38- cells were significantly higher in cultures on AFT024 than in those on HS (n=9). The effects of AFT024 on preservation of primitive progenitors were not seen in transwell (noncontact) cultures. Thus, AFT024 acts by direct contact to maintain the phenotype and function of the most primitive and quiescent human progenitors currently identifiable by in vitro assays.
PMID: 9657136, UI: 98319218
Epidermal Stem Cells
Exp Cell Res 1998 Oct 10;244(1):184-95
Selection and extended growth of murine epidermal stem cells in culture.
Bickenbach JR, Chism E
Department of Anatomy and Cell Biology, The University of Iowa, Iowa City, Iowa, 52242-1109, USA. bickenbach@uiowa.eduContinuously renewing epithelia contain small undifferentiated stem cells capable of self-renewal and maintenance of the differentiating cell population. In murine epidermis stem cells have been identified as label-retaining cells (LRCs) by long-term retention of tritiated thymidine or BrdU. It has been suggested that epidermal stem cells adhere to basement membranes through differential expression of specific integrins. To determine whether we could use a specific integrin to enrich for murine epidermal stem cells, we tested adherence of LRCs to several substrates. Regardless of the substrate used, approximately 10% of total basal cells and 100% of LRCs adhered in 10 min. In our medium specifically formulated for murine keratinocytes, rapidly adherent stem cells formed large colonies and could be used to form a structurally complete epidermis in organotypic culture. They showed a fivefold greater transient transfection efficiency than total basal cells, and when individual adherent cells were transduced with a retroviral vector, they formed large clones. Although these stem cells grew more slowly than the total basal cell population, they could be subcultured more times. Our results indicate that murine epidermal stem cells can be selected by rapid attachment to a substrate, but not by one specific integrin, and that they can be expanded in culture if the appropriate conditions are maintained. Copyright 1998 Academic Press.
PMID: 9770361, UI: 98445282
Theriogenology: January 1 2001 Vol.55 No.1, page 241
Culture in Microchannels Enhances In Vitro Embryonic Development of Preimplantation Mouse Embryos
S. Raty1, J.A. Davis1, D.J Beebe1,2 , S.L. Rodrireuz-Zas1, and M.B. Wheeler1
1University of Illinois at Urbana-Champaign, Urbana, IL, USA,2 University of Wsconsin-Madison, Madison,WI,USAValues on the same row differ at *P<0.05, or **P<0.01.
Table 1: Least Square means of the percentages of
embryos at a particular stage of development and time point.Stage of Development Microchannels (n=170) Control(droplet)(n=170) Standard error(%) 16-cell/morula @ 24hr 23.5(n=40)* 47(n=8) 6.2 Blastocysts @ 48hr 17.6(n=30)** 24(n=4) 2.4 Blastocysts @ 72hr 72.9(n=117)** 42.9(n=73) 4.6 Hatched Blastocysts @ 72hr 4.1(n=7) 0(n=0) 1.2 Hatched Blastocysts @ 96hr 26.5(n=45)** 8.8(n=15) 3.2 Degenerated Embryos @ 96hr 20.0(n=34)** 42.9(n=73) 4.6 Quotes & Notes:This was a student poster presentation at the 2001 IETS meeting, so far the abstract is unavailable through pubmed.
Comment:
This could have significant benefit throughout the entire IVF industry if the results carry over to human embryos and are repeatable.
This study essentally demonstrates the potential benefit of maintaining blastocysts in a constant stream of fresh media, with a gradual rather than abrupt transtion between G1 and G2 media, in which primary energy substrates differ. See next reference.
Theriogenology 1998 Jan 1;49(1):83-102
Changes in requirements and utilization of nutrients during mammalian preimplantation embryo development and their significance in embryo culture.
Gardner DKColorado Center for Reproductive Medicine, Denver 80110, USA.
Along with the transition from maternal to embryonic genome control the mammalian preimplantation embryo undergoes significant changes in its physiology during development. Concomitant with these changes are altering patterns of nutrient uptake and differences in the subsequent fate of such nutrients. The most significant nutrients to the developing mammalian preimplantation embryo are carbohydrates and amino acids, which serve not only to provide energy but also to maintain embryo function by preventing cellular stress induced by suboptimal culture conditions in vitro. It is subsequently proposed that optimal development of the mammalian embryo in culture requires the use of two or more media, each designed to cater for the changing requirements of the embryo. Importantly, culture conditions that maintain the early embryo are not ideal for the embryo post-compaction, and conditions that support excellent development and differentiation of the blastocyst can actually be inhibitory to the zygote. A marker of in vitro-induced cellular stress to the embryo is the relative activity of the metabolic pathways used to generate energy for development. Quantification of embryo energy metabolism may therefore serve as a valuable marker of embryo development and viability.
PMID: 10732123Comment:
An abrupt transition in the composition of energy substrates in the culture media has the potential to induce cellular stress. See preceding reference.
Hum Reprod 2000 Jan;15(1):157-64
Culture of one-cell hamster embryos with water soluble vitamins: pantothenate stimulates blastocyst production.
McKiernan SH, Bavister BDThe effects of water-soluble vitamins, singly or in combinations, on development of hamster 1-cell embryos were examined in a protein-free, chemically defined culture medium, HECM-6. Pantothenate significantly stimulated blastocyst development compared to the vitamin-free control and to every other single vitamin, except thiamine. Ascorbic acid, biotin, choline, folic acid, inositol, niacinamide, pyridoxal, riboflavin and thiamine had no detectable stimulation or inhibition on cleavage stage development or morula/blastocyst formation. When combinations of vitamins were tested, embryo development was either unchanged or significantly greater than in the control, but never significantly greater than development with pantothenate alone. A dose response to pantothenate indicated that 3 mol/l was the optimum concentration. After embryo transfer, the percentage of live fetuses recovered per 100 1-cell embryos cultured in HECM-6 plus pantothenate (now designated HECM-9) was 24%, significantly higher than the 11% recovered from 100 1-cell embryos cultured in HECM-6 alone. This is the first report to show a stimulatory effect of a single vitamin on in-vitro development of preimplantation embryos in any mammalian species.
PMID: 10611206, UI: 20079210Quotes & Notes:Pending
Comment:Pantothenate may stimulate the production of Acetyl-CoA, which is an important substrate for acetylation reactions.The acetylated form of histone protein H1 has recently been shown to be of importance in nuclear reprogramming. Histone acetylation has been correlated with transcriptional activity. See "Chromatin Remodeling" section.
The commonly used G2.2 media for IVF does contain calcium pantothenate. What concentration I do not know.Also see this reference.
Mol Reprod Dev 2000 Sep;57(1):48-54
Potent and stage-specific action of glutathione on the development of goat early embryos in vitro.
Lee CS, Koo DB, Fang N, Lee Y, Shin ST, Park CS, Lee KKThe effect of glutathione (GSH) addition on the development of 1- or 2-cell goat early embryos in vitro was examined. Embryos were collected from superovulated Korean black goat (Capra hircus aegagrus) and cultured for 6 days in synthetic oviduct fluid medium supplemented with either bovine serum albumin (BSA) or serum. Without GSH addition, almost all embryos could not develop beyond 8- to 16-cell block. However, GSH addition greatly improved in vitro development of early embryos to blastocyst stage, and its action was highly dependent on the presence and source of proteins supplemented into the culture medium. Among the protein-supplemented cultures, GSH effect was most prominent in 10% FBS-supplemented culture, in which the proportion (91%) of blastocysts developed from early embryos was much higher than that of BSA- (42-64% depending on its content) or goat serum (GS)-supplemented cultures (21%), or even than that of somatic cell-supported co-culture (60%). As well as in terms of the morphological development, mean cell number of blastocysts (185 +/- 12) developed from FBS condition was significantly higher than that of blastocysts developed from any other culture conditions and moreover comparable to that of blastocysts developed in vivo (190 +/- 9). The viability of these blastocysts was finally confirmed by their term development (6/12) from embryo transfer. To delineate action time of GSH during embryo development, GSH was treated at 1-day intervals through 6-days culture periods excepting the last day. In the GSH-treated embryos at day 3 of culture, which corresponds to the time of in vitro 8- to 16-cell block stage, the proportion of blastocyst was markedly increased up to 77% of cultured embryos and conversely that of the arrested embryos was decreased to 7%. In the embryos treated later, however, their developmental potency decreased abruptly. Therefore, these results clearly demonstrated that GSH could greatly improve the in vitro development of goat early embryos by specifically acting on the 8- to 16-cell block stage during in vitro development, suggesting that GSH may be one of the important regulators on the development of goat embryos in vivo.
Copyright 2000 Wiley-Liss, Inc.
PMID: 10954855, UI: 20413442Quotes & Notes:Pending
Comment:Seems like a reasonable addition to G1 media....
Mean cell number is the best single criterion to apply in embryo scoring.
Biol Reprod 1999 Aug;61(2):541-7
Coenzyme Q(10) in submicron-sized dispersion improves development, hatching, cell proliferation, and adenosine triphosphate content of in vitro-produced bovine embryos.
Stojkovic M, Westesen K, Zakhartchenko V, Stojkovic P, Boxhammer K, Wolf E
Coenzyme Q(10) (CoQ(10)) is an essential component of the plasma membrane ion transporter (PMIT) system and of the electron transport chain in the inner mitochondrial membrane. Because of its intrinsic functions in cell growth and energy metabolism (ATP synthesis), and its protective effects against oxidative stress, CoQ(10) is a good candidate for supporting growth of cells in culture. However, because of its quinone structure, CoQ(10) is extremely lipophilic and practically insoluble in water. We used a specific technology to prepare a submicron-sized dispersion of CoQ(10), inhibiting re-crystallization by a stabilizer. This dispersion, which exhibits a very large specific surface area for drug dissolution, was tested as a supplement for the in vitro culture of bovine embryos in a chemically defined system. The rate of early cleavage of embryos (5- to 8-cell stages) was evaluated 66 h postinsemination (hpi) and was highest in medium supplemented with 30 or 100 microM CoQ(10) (66.5 +/- 0.8% and 68.7 +/- 1.1%, respectively) and lowest in 10 microM CoQ(10) (55.3 +/- 0.8%). The proportions of oocytes developing to blastocysts by 186 hpi were 19.0 +/- 0.6% and 25.2 +/- 0.3% in medium supplemented with 10 microM and 30 microM CoQ(10), respectively, and were significantly (p < 0.001) higher than those obtained with the equivalent amounts of stabilizer (9.9 +/- 0.4% and 11.3 +/- 0.4%). In the presence of 30 microM CoQ(10), significantly (p < 0.001) more blastocysts hatched by 210 hpi than in the equivalent amount of stabilizer (31.8 +/- 1.3 vs. 8.4 +/- 2.2). Expanded blastocysts produced in the presence of 30 microM CoQ(10) had significantly (p < 0.01) more inner cell mass cells and trophectoderm cells, and a significantly (p < 0.001) increased ATP content as compared to expanded blastocysts produced in the presence of the corresponding amount of stabilizer. Our results show that noncrystalline CoQ(10) in submicron-sized dispersion supports the development and viability of bovine embryos produced in a chemically defined culture system.
PMID: 10411538, UI: 99339862Quotes & Notes:Pending
Comment:The early embryo maintains proper intracellular pH through the action of H+/Na+ antiporter, which is ATP driven.Stress to the early embryo may deplete critical ATP reserves, a plausible factor contributing to incomplete nuclear reprogramming, especially when viewed from within the conceptual framework of enhancing "chromatin fluidity".
Increasing intracellular energy stores in the form of ATP could similarly potentiate successful nuclear reprogramming, as well as enhancing blastocyst development as demonstrated in this article.
One should strongly consider adding this form of CoQ10 to all stages of cellular culture ( donor cell, early embryo, and blastocyst)
Mol Reprod Dev 1999 Feb;52(2):149-57
Pyruvate prevents peroxide-induced injury of in vitro preimplantation bovine embryos.
Morales H, Tilquin P, Rees JF, Massip A, Dessy F, Van Langendonckt AUnite des Sciences Veterinaires, Universite catholique de Louvain,Louvain-la-Neuve, Belgium.
The impact of oxidative stress on the in vitro development of bovine embryos in synthetic oviduct fluid medium (mSOF) was assessed by using H2O2 as a stress inducer. In a preliminary experiment, a chemiluminescent method was used to measure the antioxidative capacity of the mSOF culture medium. Pyruvate was the mSOF component displaying the highest H2O2 degrading ability. Essential and nonessential amino acids also significantly reduced the H2O2 concentration, whereas lactate and glutamine were ineffective. The effect on further development of a short exposure of zygotes, 9-16-cell stage embryos and blastocysts to 0 M; 10(-7) M ; 10(-6) M, and 10(-5) M H2O2 in pyruvate-free mSOF was evaluated. Developmental rates of the H2O2-treated zygotes to the 5-8-cell or blastocyst stages and survival of H2O2-treated blastocysts were reduced in a dose-dependent manner whereas the 9-16-cell embryos were unaffected by those treatments. Blastocysts treated with H2O2 also tended to have lower numbers of bisbenzimide-stained nuclei and showed increased nuclear fragmentation. Including pyruvate in the mSOF culture medium during a 10(-5) M H2O2 pulse highly reduced the H2O2 concentration as measured by chemiluminescence and improved zygote and blastocyst development, but failed to prevent blastocyst nuclei degradation. These experiments suggest that bovine embryos show developmental change in sensitivity to exogenous H2O2, the 9-16-cell embryos being more resistant than zygotes and blastocysts and that H2O2 and its toxic effects can be attenuated by including pyruvate in the medium.
PMID: 9890745
Quotes & Notes:Pending
Comment:Human embryos are commonly cultured in a two stage G1 and G2 media. G1 contains pyruvate, which at early stages is the primary energy substrate of the developing embryo. The embryonic metabolism shifts to utilize glucose as the primary energy substrate at about the blastocyst stage. Pyruvate is retained in G2.2 media.
Hum Reprod 1996 Oct;11(10):2223-9
Comparison of mouse embryo development in open and microdrop co-culture systems.
Sherbahn R, Frasor J, Radwanska E, Binor Z, Wood-Molo M, Hibner M, Mack S, Rawlins RG
Department of Obstetrics and Gynecology, Rush University Medical Center, Chicago, IL, USA.Co-culture with numerous cell lines has been shown to improve in-vitro embryo development. It is usually performed in open culture without an oil overlay, or in relatively large volumes of medium (e.g. 0.5 ml) under oil. We compared the efficacy of open and microdrop co-culture systems using human endometrial and tubal cell lines and mouse zygotes. Although the mean pH values of the media from the tubal cell cultures (both open and oil-covered) decreased significantly over 5 days of culture, this did not appear to impair embryo development. Both co-culture and microdrop culture significantly improved blastocyst and hatching blastocyst formation rates. The combination of the two techniques (microdrop and co-culture) demonstrated the highest blastocyst formation and hatching blastocyst formation rates, as well as the highest mean cell numbers in hatching blastocysts. Co-culture in a microdrop is a superior system for mouse embryo culture.
PMID: 8943534, UI: 97098918Quotes & Notes:Pending
Comment:Cell number has been shown to be one of the best predictors of blastocyst quality see here
Oocyte ActivationNature 2000 Aug 10;406(6796):633-6
NO is necessary and sufficient for egg activation at fertilization.
Kuo RC, Baxter GT, Thompson SH, Stricker SA, Patton C, Bonaventura J, Epel DNeurosciences Program, Stanford University School of Medicine, Stanford
University, California 94305, USA.The early steps that lead to the rise in calcium and egg activation at fertilization are unknown but of great interest--particularly with the advent of
in vitro fertilization techniques for treating male infertility and whole-animal cloning by nuclear transfer. This calcium rise is required for egg activation and the subsequent events of development in eggs of all species. Injection of intact sperm or sperm extracts can activate eggs, suggesting that sperm-derived factors may be involved. Here we show that nitric oxide synthase is present at high concentration and active in sperm after activation by the acrosome reaction. An increase in nitrosation within eggs is evident seconds after insemination and precedes the calcium pulse of fertilization. Microinjection of nitric oxide donors or recombinant nitric oxide synthase recapitulates events of egg activation, whereas prior injection of oxyhaemoglobin, a physiological nitric oxide scavenger, prevents egg activation after fertilization. We conclude that nitric oxide synthase and nitric-oxide-related bioactivity satisfy the primary criteria of an egg activator: they are present in an appropriate place, active at an appropriate time, and are necessary and sufficient for successful fertilization.
PMID: 10949304, UI: 20403580
Quotes & Notes:Pending
Comment:Exposure of the reconstructed SCNT oocyte to a brief pulse of NO in culture may be an efficacious method of activation, most closely simulating the natural process with minimal invasiveness and toxicity.This experiment should be repeated with mammalian embryos.
There is also the possibility that such a method will reduce Apoptosis in the embryo by inhibiting caspases. Overexposure could also induce apoptosis.
Could brief NO exposure followed quenching with addition of oxyhaemoglobin or other NO scavengers be useful?
Biochem J 1998 Nov 15;336 ( Pt 1):1-17
Arginine metabolism: nitric oxide and beyond.
Wu G, Morris SM JrDepartments of Animal Science, Medical Physiology, and Veterinary Anatomy and Public Health, and Faculty of Nutrition, Texas A&M University, College Station, TX 77843, USA. g-wu@tamu.edu
Arginine is one of the most versatile amino acids in animal cells, serving as a precursor for the synthesis not only of proteins but also of nitric oxide, urea, polyamines, proline, glutamate, creatine and agmatine. Of the enzymes that catalyse rate-controlling steps in arginine synthesis and catabolism, argininosuccinate synthase, the two arginase isoenzymes, the three nitric oxide synthase isoenzymes and arginine decarboxylase have been recognized in recent years as key factors in regulating newly identified aspects of arginine metabolism. In particular, changes in the activities of argininosuccinate synthase, the arginases, the inducible isoenzyme of nitric oxide synthase and also cationic amino acid transporters play major roles in determining the metabolic fates of arginine in health and disease, and recent studies have identified complex patterns of interaction among these enzymes. There is growing interest in the potential roles of the arginase isoenzymes as regulators of the synthesis of nitric oxide, polyamines, proline and glutamate. Physiological roles and relationships between the pathways of arginine synthesis and catabolism in vivo are complex and difficult to analyse, owing to compartmentalized expression of various enzymes at both organ (e.g. liver, sma