scholarly journals Determinants of sperm quality and fertility in domestic species

Reproduction ◽  
2007 ◽  
Vol 134 (1) ◽  
pp. 3-17 ◽  
Author(s):  
A M Petrunkina ◽  
D Waberski ◽  
A R Günzel-Apel ◽  
E Töpfer-Petersen
Keyword(s):  
Flow Cytometry ◽  
Sperm Quality ◽  
Absolute Number ◽  
Fertilization Success ◽  
Cell Counting ◽  
Computer Assisted ◽  
Timely Manner ◽  
Domestic Species ◽  
Image Area

Fertilization success cannot be attributed solely to the absolute number of vital, motile, morphologically normal spermatozoa inseminated into the female but more especially to their functional competence. A range ofin vitrotests has therefore been developed to monitor crucial aspects of sperm function: their ability to adapt to changing osmotic conditions, to bind to the oviductal epithelium, and to undergo capacitation in an appropriate and timely manner. The tests employ flow cytometry in conjunction with fluorescent techniques, electronic cell counting, and computer-assisted image area analysis. The highly quantitative analysis provided by electronic sizing and flow cytometry enables assessment of representative cell numbers in a very short time with high reproducibility. More importantly, it allows the detection of physiological heterogeneity within an ejaculate in terms of the development of cell subpopulations and enables the kinetic analysis of changes in living cell suspensions. The tests offer a promising strategy for evaluating fertility in domestic animals. The capability for volume regulation ensures that sperm recover from the tonic shocks experienced at ejaculation and during cryopreservation. Assessment of capacitationin vitroprovides valuable information on both the sperm’s ability to respond to fertilizing conditions and the sequence and rates of ongoing capacitation/destabilization processes. The monitoring of response to capacitating conditions in kinetic terms allows the sensitive and adequate detection of sperm populations expressing fertilization attributes and their ability to respond to external stimuli in a timely manner. However, subfertility is likely to be associated with a suboptimal response (i.e. too high or too low) rather than a minimal response.

111 Improvement of cat and bull sperm quality using nanotechnology as a model for wild species

2019 ◽  
Vol 31 (1) ◽  
pp. 181
Author(s):  
C. L. Durfey ◽  
T. Rowlison ◽  
C. U. Lagu ◽  
C. Sente ◽  
M. L. Khaitsa ◽  
...  
Keyword(s):  
Iron Oxide ◽  
Magnetic Nanoparticles ◽  
Wildlife Conservation ◽  
Sperm Quality ◽  
Computer Assisted ◽  
Magnetic Iron Oxide Nanoparticles ◽  
Noninvasive Technique ◽  
Domestic Species ◽  
Reproductive Techniques

Assisted reproductive techniques (ART) are widely used in domestic species, with increasing applications in wildlife conservation. However, the currently available techniques for semen preparations are not fully reliable or applicable in all species to ensure successful ART outcomes. Recent developments in nanotechnology offer new horizons for further optimization of sperm preparations. The use of conjugated magnetic nanoparticles allows for selective targeting and removal of moribund spermatozoa through the technique known as nanopurification, which has shown to be beneficial in domestic boars and bulls. Nanopurification is a rapid, straightforward, effortless, and noninvasive technique that, when applied in wildlife or endangered species, is expected to have a tremendous impact on ART outcomes. In this study, we evaluated the effectiveness of magnetic (iron oxide) nanoparticles designed to target moribund spermatozoa (e.g. acrosome reacted) on domestic felid and threatened bovid species. Fresh epididymal spermatozoa of domestic cats and ejaculated spermatozoa from genetically valuable threatened bulls (Ankole and Sahiwal) were collected and mixed with iron-oxide magnetic nanoparticles (MNP). After 30min of incubation at 38°C, semen mixtures were placed on an electromagnetic field (10-15min) to trap MNP-bound spermatozoa (moribund), followed by the elution of viable nontrapped spermatozoa (nanopurified). All samples were analysed for motility characteristics using a computer-assisted sperm analyzer. Aliquots of cat spermatozoa were subjected to (1) morphology and acrosome analyses (n=4-8 replicates), (2) cryotolerance to liquid nitrogen storage (n=4 replicates), and (3) IVF (n=1 replicate). Data were analysed with Student’s t-test and P<0.05 indicated significant differences. Regardless of the species, both control and nanopurified spermatozoa revealed comparable motility characteristics (P>0.05). Sperm MNP-bound samples exhibited higher proportions of moribund spermatozoa as compared to their nanopurified and control nonpurified counterparts (57.5v. 47.5 and 49.5%, respectively; P<0.05). Cryopreservation revealed a trend for higher post-thaw motility of nanopurified cat spermatozoa (18%) v. the controls (12%). The use of frozen-thawed control v. nanopurified cat spermatozoa to in vitro fertilize oocytes (n=13 per group) resulted in comparable embryo cleavage (15v. 31%, respectively) and blastocyst (15v. 8%, respectively) rates. This preliminary study indicates the successful interactions of MNP nanoparticles with feline and bovine spermatozoa. The nanopurification seems to improve the cryotolerance of cat spermatozoa without affecting their fertilization potential. Additional research is being conducted to confirm the current findings and optimize this novel technique for future implementation in conservation breeding programs. Work supported by USDA-ARS 58-6402-3-018 and Morris Animal Veterinary Student Scholarship - D18ZO-608.

Applications and interpretation of computer-assisted sperm analyses and sperm sorting methods in assisted breeding and comparative research

2007 ◽  
Vol 19 (6) ◽  
pp. 709 ◽  
Author(s):  
William V. Holt ◽  
Justine O'Brien ◽  
Teresa Abaigar
Keyword(s):  
Flow Cytometry ◽  
Comparative Research ◽  
Practical Knowledge ◽  
Sperm Quality ◽  
Data Interpretation ◽  
Computer Assisted ◽  
Essential Requirement ◽  
Agricultural Industry ◽  
Domestic Species ◽  
Sperm Dna

Theoretical and practical knowledge of sperm function is an essential requirement in almost every aspect of modern reproductive technology, if the overarching objective is the eventual production of live offspring. Artificial insemination (AI) techniques depend on the availability of high quality semen, whether fresh, diluted and stored, or frozen. Assessing such semen for quality and the likelihood of fertility is therefore also important, as much time, resources and effort can easily be wasted by using poor samples. Some semen technologies are aimed not at quality assessment, but at attempting to skew the breeding outcomes. Sex preselection by separating the male- and female-bearing spermatozoa using flow cytometry is now practised routinely in the agricultural industry, but speculatively it may eventually be possible to use other genetic markers besides the sex chromosomes. A moment’s reflection shows that although sex-biasing flow cytometry technology is well developed and generally fulfils its purpose if presorting of sperm quality is adequate, other technologies aimed specifically at semen assessment are also sophisticated but provide inadequate data that say little about fertility. This is especially true of instrumentation for objective sperm motility assessment. Here we aim to examine this technological paradox and suggest that although the sperm assessment equipment might be sophisticated, the shortcomings probably lie largely with inappropriate objectives and data interpretation. We also aim to review the potential value and use of sperm sexing technology for non-domestic species, arguing in this case that the limitations also lie less with the technology itself than with the applications envisaged. Finally, the potential application of a sorting method directed at motility rather than sperm DNA content is discussed.

scholarly journals Therapeutic Effect of Exogenous Treg Cells on Collagen-Induced Arthritis and Rheumatoid Arthritis

2019 ◽  
Author(s):  
Shutong Li ◽  
Hongxing Wang ◽  
Hui Wu ◽  
Guoqing Zhang ◽  
Xiaotian Chang
Keyword(s):  
Rheumatoid Arthritis ◽  
Flow Cytometry ◽  
Peripheral Blood ◽  
Synovial Fibroblast ◽  
Treg Cells ◽  
Cell Counting ◽  
Fibroblast Cells ◽  
Collagen Induced Arthritis ◽  
Gm Csf

Abstract Background Regulatory T (Treg) cells have anti-inflammatory and anti-autoimmune functions. The proportion and functions of Treg cells are perturbed in rheumatoid arthritis (RA) patients. Methods Human Treg cells were induced to amplify in vitro and cocultured with RA synovial fibroblast cells (RASFs). The proliferation and apoptosis of RASFs were determined by the cell counting kit-8 (CCK-8) assay and flow cytometry, respectively. Human Treg cells were also injected to collagen-induced arthritis (CIA) rats via the tail vein. Changes in lymphocyte subtypes and cytokines in the peripheral blood and spleen were observed by flow cytometry. Results After coculture with the Treg cells, the proliferation of RA synovial fibroblast cells decreased (p<0.01), and the rate of apoptosis increased (p=0.037). The human Treg cells were injected into the tail veins of collagen-induced arthritis (CIA) rats. The severity of the CIA was reduced (p<0.01) following the injection, the percentages of rat endogenous Treg cells in the peripheral blood and spleen increased significantly (p=0.007 and p<0.01, respectively), and the proportion of B cells decreased (p=0.031). The levels of interleukin IL-5 and IL-6 and the Th1/Th2 ratio in the peripheral blood were significantly decreased (p=0.013, 0.009 and 0.012, respectively). The number of NK cells and the levels of IL-4, IL-13, TNF-α, IFN-γ and GM-CSF in the peripheral blood and spleen did not change significantly. Conclusion These results suggest that exogenous Treg cells play a therapeutic role in RA and CIA. Treg cell treatment could serve as a therapy for RA.

scholarly journals Effect of Silicon, Titanium, and Zirconium Ion Implantation on NiTi Biocompatibility

2012 ◽  
Vol 2012 ◽  
pp. 1-16 ◽  
Author(s):  
L. L. Meisner ◽  
A. I. Lotkov ◽  
V. A. Matveeva ◽  
L. V. Artemieva ◽  
S. N. Meisner ◽  
...  
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Mesenchymal Stem Cells ◽  
Ion Implantation ◽  
Cell Counting ◽  
High Dose ◽  
Surface Layers ◽  
Test Flow ◽  
Ion Implanted

The objective of the work was to study the effect of high-dose ion implantation (HDII) of NiTi surface layers with Si Ti, or Zr, on the NiTi biocompatibility. The biocompatibility was judged from the intensity and peculiarities of proliferation of mesenchymal stem cells (MSCs) on the NiTi specimen surfaces treated by special mechanical, electrochemical, and HDII methods and differing in chemical composition, morphology, and roughness. It is shown that the ion-implanted NiTi specimens are nontoxic to rat MSCs. When cultivated with the test materials or on their surfaces, the MSCs retain the viability, adhesion, morphology, and capability for proliferationin vitro, as evidenced by cell counting in a Goryaev chamber, MTT test, flow cytometry, and light and fluorescence microscopy. The unimplanted NiTi specimens fail to stimulate MSC proliferation, and this allows the assumption of bioinertness of their surface layers. Conversely, the ion-implanted NiTi specimens reveal properties favorable for MSC proliferation on their surface.

Effect of transfection and co-incubation of bovine sperm with exogenous DNA on sperm quality and functional parameters for its use in sperm-mediated gene transfer

Zygote ◽  
2016 ◽  
Vol 25 (1) ◽  
pp. 85-97 ◽  
Author(s):  
María Elena Arias ◽  
Esther Sánchez-Villalba ◽  
Andrea Delgado ◽  
Ricardo Felmer
Keyword(s):  
Gene Transfer ◽  
Sperm Quality ◽  
Computer Assisted ◽  
Exogenous Dna ◽  
Sperm Analysis ◽  
Functional Parameters ◽  
Transgenic Embryos ◽  
Fertilization Capacity ◽  
Motility Parameters

SummarySperm-mediated gene transfer (SMGT) is based on the capacity of sperm to bind exogenous DNA and transfer it into the oocyte during fertilization. In bovines, the progress of this technology has been slow due to the poor reproducibility and efficiency of the production of transgenic embryos. The aim of the present study was to evaluate the effects of different sperm transfection systems on the quality and functional parameters of sperm. Additionally, the ability of sperm to bind and incorporate exogenous DNA was assessed. These analyses were carried out by flow cytometry and confocal fluorescence microscopy, and motility parameters were also evaluated by computer-assisted sperm analysis (CASA). Transfection was carried out using complexes of plasmid DNA with Lipofectamine, SuperFect and TurboFect for 0.5, 1, 2 or 4 h. The results showed that all of the transfection treatments promoted sperm binding and incorporation of exogenous DNA, similar to sperm incorporation of DNA alone, without affecting the viability. Nevertheless, the treatments and incubation times significantly affected the motility parameters, although no effect on the integrity of DNA or the levels of reactive oxygen species (ROS) was observed. Additionally, we observed that transfection using SuperFect and TurboFect negatively affected the acrosome integrity, and TurboFect affected the mitochondrial membrane potential of sperm. In conclusion, we demonstrated binding and incorporation of exogenous DNA by sperm after transfection and confirmed the capacity of sperm to spontaneously incorporate exogenous DNA. These findings will allow the establishment of the most appropriate method [intracytoplasmic sperm injection (ICSI) orin vitrofertilization (IVF)] of generating transgenic embryos via SMGT based on the fertilization capacity of transfected sperm.

scholarly journals Exosomes derived from HEK293T cells interact in an efficient and noninvasive manner with mammalian sperm in vitro

Nanomedicine ◽  
2020 ◽  
Vol 15 (20) ◽  
pp. 1965-1980
Author(s):  
Teresa Vilanova-Perez ◽  
Celine Jones ◽  
Stefan Balint ◽  
Rebecca Dragovic ◽  
Michael L Dustin ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Mitochondrial Membrane ◽  
Membrane Integrity ◽  
Computer Assisted ◽  
Sperm Function ◽  
Sperm Analysis ◽  
Mammalian Sperm ◽  
Boar Sperm ◽  
Computer Assisted Sperm Analysis

Aim: To investigate exosomes as a noninvasive delivery tool for mammalian sperm. Materials & Methods: Exosomes were isolated from HEK293T cells and co-incubated with boar sperm in vitro. Results: Internalized exosomes were detected within 10 min of co-incubation. Computer-assisted sperm analysis and flow cytometry demonstrated that even after 5-h of exposure to exosomes, there were no significant deleterious effects with regard to sperm motility, viability, membrane integrity and mitochondrial membrane potential (p > 0.05), thus indicating that exosomes did not interfere with basic sperm function. Conclusion: HEK293T-derived exosomes interacted with boar sperm without affecting sperm function. Exosomes represent a versatile and promising research tool for studying sperm biology and provide new options for the diagnosis and treatment of male infertility.

145 ProAKAP4 concentrations in semen as a predictive tool of bull fertility: A preliminary study

2020 ◽  
Vol 32 (2) ◽  
pp. 199 ◽  
Author(s):  
I. Ruelle ◽  
N. Seregeant ◽  
D. Bencharif ◽  
F. Charreaux ◽  
C. Thorin ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Sperm Quality ◽  
Intrauterine Insemination ◽  
Human Reproduction ◽  
Sperm Parameters ◽  
Enzyme Linked Immunosorbent Assay ◽  
Computer Assisted ◽  
Predictive Tool ◽  
Progressive Motility ◽  
Preliminary Study

Recently, ProAKAP4 has been described as a pertinent indicator of sperm quality in humans, pigs, and stallions. In knockout mouse models lacking AKAP4 expression, the male mice were infertile. As high proAKAP4 levels were significantly correlated with a lower proportion of abortions in intrauterine insemination settings in human reproduction, proAKAP4 could be considered a pertinent new sperm parameter for assessing embryo quality. Our main goal was to assess the proAKAP4 concentrations in Holstein bull semen for comparison with the motility sperm parameters and fertility outcomes in post-thawed conditions. Straws issued from 52 ejaculates from 13 bulls, retrospectively identified with known nonreturn rates (NRR) as a fertility indicator, were provided by Evolution XY. Expression of ProAKAP4 and AKAP4 was assessed using enzyme-linked immunosorbent assay, western blotting, flow cytometry, and microscopy methods. Using the Bull 4MID kit (4BioDx), striking variations in proAKAP4 concentrations were observed independently of the classic sperm parameters that were measured using computer-assisted semen analysis. A mean proAKAP4 concentration of 44.42ng per 10 million spermatozoa was obtained through all our series. Interestingly, the variations in proAKAP4 concentrations were positively correlated with progressive motility and with the linearity coefficient parameter. Furthermore, the post-thawed concentrations of proAKAP4 were significantly higher in bulls with a higher NRR in a field study of more than 190 000 AI. We then demonstrated for the first time a correlation between the semen concentration of proAKAP4 and NRR (P=0.05) in bulls. Threshold values of proAKAP4 were then determined, with good values being between 25 and 60ngmL−1. Below 25ngmL−1, the sperm were of poor quality. The proportion of functional spermatozoa (i.e. spermatozoa expressing proAKAP4 in ejaculates) was assessed using flow cytometry. We observed that the cell debris and dead spermatozoa were never immunolabeled with proAKAP4 antibodies. On testis tissue sections, proAKAP4 was expressed only from the spermatids stages up to the ejaculated spermatozoa, being influenced by external factors and reflecting good spermatogenesis. Our preliminary study highlighted the pertinence of proAKAP4 in assessing sperm quality in bulls. It could be interesting to further analyse the effect of proAKAP4 level of expression on capacitation and IVF. As high levels of proAKAP4 were significantly correlated with fertility rates and with progressive motility, proAKAP4 could be proposed as a predictive marker of bull fertility and could be further investigated to evaluate the quality of invitro-produced embryos.

53 FREEZING BULL SEMEN IN A SYNTHETIC MEDIUM

2017 ◽  
Vol 29 (1) ◽  
pp. 134
Author(s):  
L. Gavin-Plagne ◽  
P. Bodranghien ◽  
A. Vachet ◽  
L. Commin ◽  
S. Buff ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Synthetic Medium ◽  
Calf Serum ◽  
Egg Yolk ◽  
Wilcoxon Test ◽  
Computer Assisted ◽  
Sperm Cells ◽  
Bull Semen ◽  
Significant Difference

Animal-derived products are currently used to cryopreserve sperm cells. However, these products represent potential risks of contamination by pathogens. Optidyl® (IMV Technologies, L’Aigle, France), containing egg yolk, is a reference product in Europe used routinely in bovine insemination centers. Commercial media such as soy lecithin or liposome-based media have been used to replace extenders containing products derived from animals. However, their protective effect could be called into question because of their non-synthetic or unstable properties. Despite these innovative extenders on the market, it might be necessary for sanitary reasons to cryopreserve bull semen in a stable and synthetic extender. CRYO3 (Stem Alpha, Saint-Genis l’Argentière, France), a serum-free and protein-free medium used for cryopreserving somatic and stem human cells, is a potential medium to cryopreserve reproductive cells. Recently, CRYO3 improved cryopreservation of in vitro-produced bovine embryos compared with fetal calf serum and BSA-based media. Thus, it could be interesting to test this medium on sperm cells. The objective of this study was to compare 2 in vitro freezing media on bull semen: a commercial egg yolk-based medium and a synthetic medium containing 20% CRYO3. Sperm from 5 bulls were collected in Auriva station (Brindas, France). A sample of each ejaculate was used to assess fresh semen quality. The remaining sperm of each bull was split and diluted in 2 media: Optidyl® and a CRYO3-based medium. Semen was equilibrated at least 4 h at 4°C before being packaged in 0.25-mL French straws, and then frozen into a programmable freezer and plunged into liquid nitrogen. Osmolarity and pH were respectively 1462 mOsm/kg and 6.9 for Optidyl® and 1286 mOsm/kg and 6.8 for CRYO3 medium. Viability (with SYBR-14 and propidium iodide) and high mitochondrial membrane potential (hMMP) (with JC-1) were assessed using flow cytometry. A hypo-osmotic swelling test was performed to assess functional membrane integrity (FMI). The motility parameters were evaluated by computer-assisted sperm analysis. Statistical analysis was performed using Wilcoxon test with the R software. Fresh sperm showed 52% viability, 64% hMMP, 74% FMI, and 62 and 76% progressive and total motility, respectively. For all parameters measured, no significant difference was observed between extenders and between fresh and frozen–thawed sperm (P > 0.05). However, Optidyl® showed clearly better survival than CRYO3 (45% v. 16% for viability, 58% v. 20% for hMMP, 67% v. 25% for FMI, 51% v. 14% for progressive motility and 72% v. 32% for total motility, respectively). These results show that it is possible to freeze bovine semen in synthetic extender though the low survival rate after freezing-thawing. Indeed, it is known that motility and flow cytometry parameters are not necessarily good indicators of fertility. Artificial inseminations will be done to verify the fertility of the sperm cryopreserved in CRYO3. Repetitions with more bulls from different breeds will be performed to complete this preliminary work. This work was supported by grant CRB-ANIM ANR-11-INBS-0003.

180 ASSESSMENT OF BULL SEMEN QUALITY LOADED IN NEW SensiTemp STRAWS USING SEMEN AND IN VITRO PRODUCTION TECHNOLOGIES

2016 ◽  
Vol 28 (2) ◽  
pp. 221
Author(s):  
D. Le Bourhis ◽  
S. Camugli ◽  
P. Salvetti ◽  
L. Schibler ◽  
E. Schmitt
Keyword(s):  
Sperm Motility ◽  
Semen Quality ◽  
Sperm Quality ◽  
Semen Analysis ◽  
Membrane Integrity ◽  
Computer Assisted ◽  
Cleavage Rate ◽  
Fluid Medium ◽  
And Control

SensiTemp, a new in vitro maturation (IMV) bull straw concept, presents the advantage of colour changing while the straw is thawed. The colour of frozen straws is blue and straws start to become white when the temperature reaches 33°C, with a complete change of colour at 37°C. The objective of this study is to assess sperm quality after thawing of semen frozen in SensiTemp from 2 bulls, by analysing, in experiment 1, sperm motility and membrane integrity using computer-assisted semen analysis (CASA) and flow cytometry (FC), and, in experiment 2, the in vitro embryo production (IVP) using IVP technologies [IVM, IVF, and in vitro culture (IVC)]. The ejaculates of 2 bulls, selected during preliminary experiments on high in vitro fertility, were harvested at CIA L’Aigle, France, and split ejaculates were frozen in experimental (SensiTemp) and conventional (control) straws. In experiment 1 after thawing semen from the 2 types of straws (5 pooled straws each; 2 replicates), motility was assessed using the IVOS CASA system (Hamilton Thorne Inc., Beverly, MA, USA) and membrane integrity was evaluated through FC with Cytosoft software (Millipore-Guava Technologies Inc., Hayward, CA, USA). In experiment 2, IVF was used to evaluate the non-toxicity of SensiTemp and control straws. Cumulus-oocyte complexes (COC; n = 1178; 4 replicates) collected from slaughterhouse ovaries were matured in IVM medium (TCM-199 with bicarbonate, Sigma-Aldrich, Saint Quentin Fallavier, France; 10 µg mL–1 FSH-LH, Reprobiol, Liège, Belgium; and 10% FCS, Thermo Fisher, Illkirch, France) for 22 h. After fertilization, presumptive zygotes of each group (SensiTemp and control for each bull) were cultured in synthetic oviduct fluid medium (SOF, Minitube, Tiefenbach, Germany) with 1% estrous cow serum (ECS) and 0.6% BSA (Sigma-Aldrich, France) up to 8 days. All cultures were conducted at 38.5C in 5% CO2, and 5% O2. The cleavage and blastocysts rates were evaluated on Days 3 and 7, respectively, for each group. Embryo quality was recorded on Day 7 according to the IETS evaluation. Data from each bull were analysed separately using the chi-squared test (P < 0.05). In experiment 1, neither sperm motility from bull 1 (61.2 and 60.5%) and bull 2 (66.2 and 66.5%) nor membrane integrity from bull 1 (58.6 and 52.2%) and bull 2 (61.0 and 61.9%) were different between SensiTemp and control, respectively. Results from experiment 2 showed no difference (P > 0.05) in cleavage rate between SensiTemp and control for the 2 bulls: 92.1 and 91.7% for bull 1 and 94.2 and 94.6% for bull 2 respectively. The blastocysts rate on Day 7 did not differ (P > 0.05) among groups (47.5, 47.1 and 51.3, 50.4% for SensiTemp and control bull 1 and bull 2, respectively) nor the quality of embryos retrieved in the different groups: 25.4, 23.3, and 30.8, 29.6% in grade 1 embryo for SensiTemp and control bull 1 and bull 2, respectively. Those results demonstrate, in vitro, that the new SensiTemp straws were non-toxic and did not affect the semen quality after thawing nor did the SensiTemp straws affect the ability of sperm cells to fertilize oocytes and produce 8-day-old embryos.

Dysfunction of Bone Marrow Endothelial Progenitor Cells from Subjects with Poor Graft Function Following Allogeneic Hematopoietic Stem Cell Transplantation Can be Improved By Atorvastatin

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3386-3386
Author(s):  
Minmin Shi ◽  
Yuan Kong ◽  
Yang Song ◽  
Yuqian Sun ◽  
Yu Wang ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Mapk Pathway ◽  
Graft Function ◽  
Cell Counting ◽  
Hematopoietic Stem ◽  
Atorvastatin Treatment ◽  
Poor Graft Function ◽  
And Function

Abstract Background Poor graft function (PGF) is a serious complication post allo-HSCT. The definition is a hypo- or aplastic bone marrow with 2 or 3 of the following: (1) neutrophils ≤0.5×10E+9/L; (2) platelets ≤20×10E+9/L; and/or (3) hemoglobin concentration ≤70 g/L for at least 3 consecutive days after day +28 post-HSCT. The mechanisms of PGF remain poorly understood. Murine studies suggest that endothelial progenitor cells (EPCs) are preferential supporting cells for hematopoietic stem cells (HSCs) in the bone marrow (BM) microenvironment. In addition, our previous prospective, nested case-control study found that a reduced number of BM EPCs was an independent risk factor for the occurrence of PGF after allo-HSCT. However, little is known about the functional role of BM EPCs and how to improve impaired BM EPCs in PGF. Atorvastatin is widely used in the treatment of dyslipidemia and associated vascular abnormalities. Furthermore, atorvastatin has been reported to improve the mobilization and function of circulating EPCs in a number of diseases, such as heart disease, diabetes and others. Nevertheless, no previous studies have focused on the roles of atorvastatin on bone marrow-derived EPCs in subjects with poor graft function following allo-HSCT. Aims To evaluate the function of BM EPCs in subjects with PGF. Moreover, to investigate the effect of atorvastatin on the number and function of cultivated BM EPCs derived from subjects with PGF and its underling molecular mechanisms. Methods Three cohorts were included: subjects with PGF (N=23), subjects with good graft function (N=23), defined as persistent successful engraftment after allotransplant, and transplant donors as normal controls (N=23). Atorvastatin (0.5nM,50nM,500nM) was administrated to the 5 day cultivated BM EPCs from subjects with PGF until tested on day 7. The number and functions of CD34(+)/CD133(+)/KDR(+)EPCs were evaluated by flow cytometry, cell counting, DiI-Ac-LDL and FITC-lectin-UEA-1 double staining, migration, tube formation test and apoptosis. Reactive Oxygen Species (ROS) level was evaluated by flow cytometry. Cell proliferation was determined by cell counting kit-8 assay. Protein expression for ERK,JNK, p38, Akt was measured by flow cytometry and western blots. EPCs-CD34+ co-culture system and Colony-forming unit (CFU) assays were used to evaluate the supporting effect that atorvastatin exposure on hematopoietic progenitors in vitro. Results Human BM EPCs was characterized by the spindle shape and expression of CD34, CD309 and CD133 at day 7 of cultivation. Dysfunctional BM EPCs, which were characterized by impaired proliferation, migration, angiogenesis, and higher levels of reactive oxygen species and apoptosis, were revealed in subjects with PGF. Activation of p38 and its downstream transcription factor cAMP-responsive element-binding protein (CREB) were detected in BM EPCs from subjects with PGF. Furthermore, the number and function of BM EPCs derived from subjects with PGF were enhanced by atorvastatin treatment in vitro through down-regulation of the p38 mitogen-activated protein kinase (MAPK) pathway. The colony-forming unit plating efficiency in CD34+ BM cells was also improved by atorvastatin when co-cultured with BM EPCs from subjects with PGF. Atorvastatin demonstrated similar improvement effects on the number and function of bone marrow EPCs as ROS inhibitor and p38 inhibitor from subjects with PGF. Conclusion In summary, dysfunctional BM EPCs were observed in subjects with PGF. Atorvastatin treatment in vitro quantitatively and functionally improved BM EPCs derived from subjects with PGF through down-regulation of the p38 MAPK pathway. These data indicate that atorvastatin represents a promising therapeutic approach for repairing impaired BM EPCs in subjects with PGF post-allotransplant. Disclosures No relevant conflicts of interest to declare.

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