A Modified Handmade Cloning Method for Dromedary Camels.

Camels play very important economic and sociocultural roles for communities residing in arid and semi-arid countries. The positive impacts of cloning on genetic gain in camel species are indisputable, considering the unique ability of cloning to produce a large number of offspring of a predefined sex and genotype using somatic cells obtained from elite animals, live or dead, and within any age category. However, the current low efficiency of camel cloning seriously limits its commercial applicability. We have systematically optimized technical and biological factors for dromedary camel cloning. In this chapter, we present the details of our current standard operating procedure for dromedary camel cloning, namely, "modified handmade cloning (mHMC)."

Vitrification of camel oocytes transiently impacts mitochondrial functions without affecting the developmental potential after intracytoplasmic sperm injection and parthenogenetic activation.

Oocyte vitrification preserves the female genetic resources of elite dromedary camels. In the current study, we aimed to explore the effects of vitrification of camel oocytes on mitochondrial activity, redox stress, and expression of genes related to mitochondrial function, apoptosis, pluripotency, and cytoskeleton. Moreover, we investigated developmental competence of vitrified oocytes after parthenogenetic activation. Oocytes vitrified with the Cryotop method were compared with the fresh oocytes. Our results showed that vitrification led to increased ROS production in oocytes as evidenced by an increase in the DCFDHA fluorescence intensity, and lower mitochondrial activity. At the molecular level, vitrification reduced mRNA expression of many genes, including those related to mitochondrial function (TFAM, MT-CO1, MFN1, ATP1A1, NRF1), pluripotency (SOX2 and POU5F1), and apoptosis (p53 and BAX). In contrast, expression of KLF4 and cytoskeleton-related genes (ACTB and KRT8) was not affected. However, we found no difference in the rates of oocyte survival, cleavage, and blastocyst development, and blastocyst hatching between fresh and vitrified oocytes after warming. Our results indicate that although vitrification of camel metaphase II (MII) oocytes adversely affected mitochondrial functions, the effect was transient without compromising the developmental potential of the oocytes after parthenogenetic activation.

Survival, re-expansion, and pregnancy outcome following vitrification of dromedary camel cloned blastocysts: A possible role of vitrification in improving clone pregnancy rate by weeding out poor competent embryos.

There is a clinical demand for efficient cryopreservation of cloned camel embryos with considerable logistic and economic advantage. Vitrification of in vivo derived embryos has been reported in camels, but there is no study on vitrification of cloned embryos. Moreover, whether characteristic differences between cloned and in vivo derived embryos imply different vitrification requirement is unresolved. Here, we compared survival, re-expansion and pregnancy rates of cloned embryos vitrified using two commercial vitrification kits (Cryotec and Kitazato), developed basically for human embryos, and a vitrification protocol developed for in vivo camel embryos (CVP). Cloned embryos responded dynamically to vitrification-warming steps in commercial kits, with a flat shrinkage in the final vitrification solution and a quick re-expansion to the original volume immediately after transferring to the isotonic warming solution. Contrarily, full shrinkage was not observed in CVP method, and majority of embryos were still collapsed post-warming. The immediate re-expansion was highly associated and predictive of higher survival and total cell number, and also better redox state of embryos vitrified by Cryotec and Kitazato kits compared to CVP method. Importantly, while 30% blastomere loss, verified by differential dye exclusion test, was tolerated in vitrified embryos, >50% blastomeres loss in non-expanded blastocysts implied the minimal essential cell survival rate for blastocoelic cavity re-expansion in vitrified cloned camel blastocysts, irrespective of vitrification method. A protocol-based exposure of embryos to cryoprotectants indicated that cryoprotectant toxicity, per se, may not be involved in lower cryosurvival of embryos in CVP vs. Cryotec and Kitazato. The initial pregnancy rates were numerically higher in Cryotec and Kitazato frozen transfers compared to fresh transfer (56.3, 60 and 33.3%, respectively), and importantly, a higher percentage of established pregnancies in vitrified groups passed the critical 3 months period of early embryonic loss compared to sibling fresh clone pregnancies (50, 40, and 10%, respectively). Results confirmed the suitability of Cryotec and Kitazato kits for vitrification of cloned camel embryos and that vitrification may improve pregnancy outcome by weeding out poor competent embryos.

Development of a modified method of handmade cloning in dromedary camel.

In this study, a modified method of handmade cloning (m-HMC), which had been originally developed in sheep, was used for somatic cell nuclear transfer (SCNT) in the dromedary camel. The unique feature of m-HMC over current SCNT methods lies in the use of a simple device (a finely drawn micropipette made of Pasteur pipette) for chemically-assisted enucleation of oocytes under a stereomicroscope with improved efficiency and ease of operation. Using this system, the throughput of cloned embryo reconstitution was increased over 2-fold compared to the control SCNT method (c-NT). Stepwise measurement of reactive oxygen species (ROS) revealed that method, steps, and duration of SCNT all influenced oxidative activity of oocytes, but their impact were not similar. Specifically, UV-assisted oocyte enucleation was identified as the major source of ROS production, which explained significantly higher total ROS of reconstituted embryos in c-NT compared to m-HMC. Fusion efficiency (95.3±3.3 vs. 75.4±7.6%) and total efficiency of blastocyst development (22.5±3.0 vs. 14.1±4.3%) were significantly higher in m-HMC compared to c-NT, respectively, and blastocysts of transferable quality were obtained in similar rates (41.9±8.2 vs. 48.0±15.2%, respectively). Significance differences were observed in total cell number (155.3±13.6 vs. 123.6±19.5) and trophectoderm (145±9.5 vs. 114.3±15.2), but not inner cell mass (10.3±4.1 vs. 9.3±5.3) counts between blastocysts developed in c-NT compared to m-HMC, respectively. However, expression of key developmental genes (POU5F1, KLF4, SOX2, MYC, and CDX2) was comparable between blastocysts of both groups. The introduced m-HMC method might be a viable approach for efficient production of dromedary camel clones for research and commercial utilization.

Evidence of Oocyte Polarity in Bovine; Implications for Intracytoplasmic Sperm Injection and Somatic Cell Nuclear Transfer.

OBJECTIVES: We recently demonstrated spatial regionalization of maternal transcripts and proteins within unfertilized ovine oocyte. Here, we investigated the likelihood of oocyte polarity for the first time in bovine. MATERIALS AND METHODS: In this experimental study, in vitro matured bovine oocytes were used for manual bisection [into oocyte halve that were near-to (HNS) and far-from (FS) spindle] or trisection [into MII-spindle (S), the spindle-side half (NS), and the distal half unassociated with the spindle (FS)]. Prepared pools of oocyte substructures were used for comparative quantitative real-time polymerase chain reaction (RT-qPCR). To map the possible preferential sperm entry point (SEP), the spatial relationship between SEP and MII-spindle was measured 5 hours post-fertilization. RESULTS: The proportional amount of maternal mRNA in S oocyte fragment was estimated to be 6 to 11-fold higher than NS and FS counterparts. The relative abundances of Nanog, Oct4, Fgf4 and Tead4 were significantly higher in HNS oocyte fragment compared t0 FS. The relative abundances of Ctnb, Carm1, Rex1, Sox2 and Cdx2 were comparable between HNS and NS oocyte fragments. FS oocyte fragment possessed significantly higher transcripts of Gata4 compared to HNS. The distribution of certain transcripts related to pluripotency and lineage commitment were different depending upon the region of the oocyte; either enriched at S (Tead4, Nanog, Ctnb and Sox2), NS (Oct4), or FS (Gata6). The SEP in almost (90%) fertilized oocytes was located in MII-hemisphere. CONCLUSIONS: The observation of spatial restriction of mRNAs and SEP within MII-oocyte may indicate that the principal forces of oocyte polarity are evolutionary conserved. This may in turn highlight the need for refinements in the methodology of intracytoplasmic sperm injection (where a sperm is injected far from the MII-spindle) and somatic cell nuclear transfer (where a major amount of regulative mRNAs that are associated with MIIspindle is removed during enucleation).

The Principal Forces of Oocyte Polarity Are Evolutionary Conserved but May Not Affect the Contribution of the First Two Blastomeres to the Blastocyst Development in Mammals.

Oocyte polarity and embryonic patterning are well-established features of development in lower species. Whether a similar form of pre-patterning exists in mammals is currently under hot debate in mice. This study investigated this issue for the first time in ovine as a large mammal model. Microsurgical trisection of unfertilized MII-oocytes revealed that cortical cytoplasm around spindle (S) contained significant amounts of total maternal mRNAs and proteins compared to matched cytoplast hemispheres that were located either near (NS) or far (FS) -to-spindle. RT-qPCR provided striking examples of maternal mRNA localized to subcellular substructures S (NPM2, GMNN, H19, PCAF, DNMT3A, DNMT1, and STELLA), NS (SOX2, NANOG, POU5F1, and TET1), and FS (GCN) of MII oocyte. Immunoblotting revealed that specific maternal proteins DNMT3A and NANOG were asymmetrically enriched in MII-spindle-half of the oocytes. Topological analysis of sperm entry point (SEP) revealed that sperm preferentially entered via the MII-spindle-half of the oocytes. Even though, the topological position of first cleavage plane with regard to SEP was quite stochastic. Spatial comparison of lipid content revealed symmetrical distribution of lipids between 2-cell blastomeres. Lineage tracing using Dil, a fluorescent dye, revealed that while the progeny of leading blastomere of 2-cell embryos contributed to more cells in the developed blastocysts compared to lagging counterpart, the contributions of leading and lagging blastomeres to the embryonic-abembryonic parts of the developed blastocysts were almost unbiased. And finally, separated sister blastomeres of 2-cell embryos had an overall similar probability to arrest at any stage before the blastocyst (2-cell, 4-cell, 8-cell, and morula) or to achieve the blastocyst stage. It was concluded that the localization of maternal mRNAs and proteins at the spindle are evolutionarily conserved between mammals unfertilized ovine oocyte could be considered polar with respect to the spatial regionalization of maternal transcripts and proteins. Even though, the principal forces of this definitive oocyte polarity may not persist during embryonic cleavages.

Epigenetic modification with trichostatin A does not correct specific errors of somatic cell nuclear transfer at the transcriptomic level; highlighting the non-random nature of oocyte-mediated reprogramming errors.

BACKGROUND: The limited duration and compromised efficiency of oocyte-mediated reprogramming, which occurs during the early hours following somatic cell nuclear transfer (SCNT), may significantly interfere with epigenetic reprogramming, contributing to the high incidence of ill/fatal transcriptional phenotypes and physiological anomalies occurring later during pre- and post-implantation events. A potent histone deacetylase inhibitor, trichostatin A (TSA), was used to understand the effects of assisted epigenetic modifications on transcriptional profiles of SCNT blastocysts and to identify specific or categories of genes affected. RESULTS: TSA improved the yield and quality of in vitro embryo development compared to control (CTR-NT). Significance analysis of microarray results revealed that of 37,238 targeted gene transcripts represented on the microarray slide, a relatively small number of genes were differentially expressed in CTR-NT (1592 = 4.3 %) and TSA-NT (1907 = 5.1 %) compared to IVF embryos. For both SCNT groups, the majority of downregulated and more than half of upregulated genes were common and as much as 15 % of all deregulated transcripts were located on chromosome X. Correspondence analysis clustered CTR-NT and IVF transcriptomes close together regardless of the embryo production method, whereas TSA changed SCNT transcriptome to a very clearly separated cluster. Ontological classification of deregulated genes using IPA uncovered a variety of functional categories similarly affected in both SCNT groups with a preponderance of genes required for biological processes. Examination of genes involved in different canonical pathways revealed that the WNT and FGF pathways were similarly affected in both SCNT groups. Although TSA markedly changed epigenetic reprogramming of donor cells (DNA-methylation, H3K9 acetylation), reconstituted oocytes (5mC, 5hmC), and blastocysts (DNA-methylation, H3K9 acetylation), these changes did not recapitulate parallel marked changes in chromatin remodeling, and nascent mRNA and OCT4-EGFP expression of TSA-NT vs. CRT-NT embryos. CONCLUSIONS: The results obtained suggest that despite the extensive reprogramming of donor cells that occurred by the blastocyst stage, SCNT-specific errors are of a non-random nature in bovine and are not responsive to epigenetic modifications by TSA.

Initial clinical outcomes of intracoronary infusion of autologous progenitor cells in patients with acute myocardial infarction.

BACKGROUND: Myocardial infarction (MI) is an irreversible cardiomyocytes injury which begins after 15-20 minutes of coronary artery occlusion. The extent of infarction is modulated by a number of factors including collateral blood supplies, medications, and ischemic preconditioning. Although angioplasty and thrombolytic agents can relieve the cause of the infarction, the time from the occlusion onset to reperfusion determines the degree of irreversible myocardial injury. Experimental studies suggested that stem cells and progenitor cells derived from bone marrow can be used in the repair of cardiac tissue after acute MI. This study was designed to investigate the feasibility, safety and initial clinical outcome of intracoronary infusion of autologous progenitor cells in patients with acute MI. METHODS: Patients with a history of anterior MI and a left ventricular ejection fraction (LVEF) less than 35 % who were candidates for coronary angioplasty were randomly allocated in a 1:1 ratio to either control or bone marrow cell groups (each including 16 patients). Thallium scan and 17-segment echocardiography analysis for regional wall motion abnormality were performed before and 1 and 6 months after intracoronary infusion of bone marrow cells. The same tests were also conducted for the control group at identical time intervals. Quantitative variables were compared by independent t-test and paired t-test. Statistical significance was assumed at a value of P < 0.05. RESULTS: LVEF in the case and control groups increased to 39.37 ± 2.47% and 31.00 ± 1.87%, respectively (P = 0.069 and 0.1, respectively). Wall motion abnormality index (WMAI) decreased insignificantly in both groups. Perfusion defect scores (PDSs) decreased significantly in the case group. CONCLUSION: In this study, autologous mesenchymal stem cell transplantation by intracoronary catheter during angioplasty in patients with a history of severe LV dysfunction caused mild increases in LVEF.

Enucleated ovine oocyte supports human somatic cells reprogramming back to the embryonic stage.

Increased possibility of universality of ooplasmic reprogramming factors resulted in a parallel increased interest to use interspecies somatic cell nuclear transfer (iSCNT) to address basic questions of developmental biology and to improve the feasibility of cell therapy. In this study, the interactions between human somatic cells and ovine oocytes were investigated. Nuclear remodeling events were first observed 3 h post-iSCNT as nuclear swelling, chromosome condensation, and spindle formation. A time-dependent decrease in maturation promoting activity of inactivated reconstructs coincided with increased aberrations in chromosome and spindle organization of the newly developed embryos. The sequence and duration of nuclear remodeling events were irrespective of donor cell type used. Although the majority of the reconstituted embryos arrested before embryonic genome activation (8-16-cell) stage, less than 5% of them could progress beyond transcription-requiring developmental stage and formed blastocyst-like structures with distinct inner cell mass and trophectoderm at days 7 and 8 post-SCNT. Importantly, real-time assessment of three developmentally important genes (Oct4, Sox2, and Nanog) indicated their upregulation in iSCNT blastocysts. Blastocyst-derived outgrowths had alkaline phosphatase activity that was lost upon passage. Collectively, this study introduced ovine oocyte as a credible cytoplast for remodeling and reprogramming of human somatic cells back to the embryonic stage and provided a platform for further studies to unravel possible differences exist between reprogramming ability of oocytes of different mammalian species.

Effect of ovarian cyclic status on in vitro embryo production in cattle.

BACKGROUND: The relationship between cyclic status of cattle ovaries on in vitro embryo development up to the blastocyst stage was investigated. MATERIALS AND METHODS: Cattle ovaries were collected immediately after slaughter and divided into three categories based on their cyclic status, which included: 1. the presence of a large follicle (LF), 2. the presence of a corpus luteum (CL) and 3. ovaries without LF or CL (WLCF). Oocytes of these ovaries were obtained and used for in vitro maturation and fertilization. Presumptive zygotes were then cultured up to the blastocyst stage in synthetic oviductal fluid culture medium. RESULTS: There were no significant differences between cleavage rates of the three groups. The rate of embryos in the compact morula stage for the CL group was 48.2% which was significantly higher than the related rate of the LF group (36.6%), but non-significantly higher than that of the ST group (45.7%). The highest blastocyst rate belonged to the CL group (54.6%) which was significantly greater than the WLCF group (32.9%) and non-significantly higher than the LF group (52.4%). There was no significant difference in blastocyst rates in the CL and LF groups. CONCLUSION: Preselection of oocyte donor ovaries containing a CL or LF can be used as a feasible and non- invasive criterion to obtain the most competent oocytes capable of development to the blastocyst stage.

Treatment of full thickness cartilage defects in human knees with Autologous Chondrocyte Transplantation.

BACKGROUND: Although a variety of strategies have been employed for managing articular cartilage defects in the knee, overall outcomes have not been satisfactory. An alternative option may be autologous chondrocyte transplantation (ACT). However, as this method is still under investigation, here we assessed the efficacy of ACT for human knee defect cartilage repair. METHODS: In a randomized clinical trial study, eleven patients (mean age 31.09 years) were enrolled in the study with full thickness cartilage defects in the knee. Arthroscopically, healthy cartilage was obtained, chondrocytes expanded for 2-3 weeks and ACT performed. Clinical status was evaluated before ACT, 6 and 12 months after ACT using the Brittberg-Peterson functional assessment and modified Cincinnati rating score. Magnetic resonance imaging (MRI) findings were evaluated based on the scoring systems used by Sally Roberts and by Henderson. RESULTS: Modified Cincinnati rating indicated significant improvement of clinical score before ACT compared to 6 (p = 0.000) and 12 (p = 0.000) months after ACT (from 2.73 before ACT to 7.27, 8.36 and 9.5 at 6, 12, and 48 months after ACT, respectively). Brittberg-Peterson functional assessment indicated a decline from 79.27 to 25.82 and 19.27 at 6 and 12 months post ACT. Further, statistical test demonstrated significant differences 6, 12 and 48 months post ACT (p = 0.007). Evaluation of MRI revealed a score of 6.5 for Henderson criteria and a score of 2.5 for Robert criteria. CONCLUSIONS: Our study demonstrated that ACT of the knee provides an excellent treatment for full thickness cartilage defects with outstanding clinical and radiological outcomes.

Soft tissue augmentation by autologous cultured fibroblasts transplantation for treatment of wrinkles and scars: a case series of 20 patients.

BACKGROUND: There are many filler agents for augmentation of static wrinkles and atrophic scars from synthetic, bio-synthetic, cadaver, animal and human sources. METHODS: The current study presents 20 patients whose facial wrinkles and lines were treated by transplantation of autologous cultured fibroblasts. The fibroblast nature of cells was confirmed by immune-staining and flow cytometry. RESULTS: The mean of improvement for this procedure at the 6 month follow up was 41%. CONCLUSIONS: In conclusion autologous fibroblast transplantation can be an effective procedure for correction of wrinkles and atrophic scars.

Can Vero cell co-culture improve in-vitro maturation of bovine oocytes?

This study was carried out to evaluate the effect of Vero cell co-culture on developmental competence of immature oocytes. Bovine cumulus-oocyte complexes (COC) were matured in presence or absence of Vero cells. Matured oocytes were inseminated and cultured for up to 9 days. Cleavage percentages were recorded on day 2 after insemination and embryos were evaluated on a daily basis. Expanding/expanded and hatching/hatched blastocysts were used for cell number assay. Results indicated a significantly greater cleavage percentage in oocytes matured in presence of Vero cells than control (86% versus 76%, P < or = 0.05). The percentages of advanced embryos appear to be greater on a daily basis in COC matured in presence of Vero cells compared with control. However, these differences were not significant. Blastocysts derived from COC matured in the presence of Vero cells had a significantly higher (P < or = 0.05) number of inner cell mass, trophectoderm and total cell number in expanding/expanded (65.25, 224.5 and 289.7 respectively) and hatching/hatched (67.75, 289.75 and 357.5) embryos in comparison to the control (42, 203.5, 245.5 and 51.3, 265, 316.3 respectively). Results confirm that co-culture of bovine COC during in-vitro maturation, enhances their ability for cleavage and for producing blastocysts with higher quality.

Effect of sperm DNA damage and sperm protamine deficiency on fertilization and embryo development post-ICSI.

The aim of this study was to evaluate the effect of sperm DNA damage and protamine deficiency on fertilization and embryo development post-intracytoplasmic sperm injection (ICSI), and also to assess the effect of protamine deficiency on DNA damage. Semen samples were collected from 28 patients participating in the ICSI programme. Following sperm preparation and ICSI, the remaining processed semen samples were used to assess protamine deficiency and DNA damage employing chromomycin A3 (CMA3) staining and comet assay, respectively. Comet parameters, CMA3 percentage positivity, fertilization rate, embryo cleavage score and embryo quality score were assessed. Except for CMA3, none of the comet parameters showed significant correlation with fertilization rate. However, among comet parameters, head area and head intensity showed positive correlation with the embryo cleavage score, while comet mean intensity and head mean intensity showed a significant negative correlation with CMA3 positivity. Results of this study demonstrate that DNA fragmentation is more frequent in protamine-deficient spermatozoa. Unlike protamine deficiency, sperm DNA fragmentation does not preclude fertilization. Nonetheless, embryos derived from spermatozoa with high DNA damage have a lower potential to reach blastocyst stage.