Publications by authors named "Cansu Agca"

39 Publications

Cryopreservation and Transplantation of Laboratory Rodent Ovarian Tissue for Genome Banking and Biomedical Research.

Methods Mol Biol 2021 ;2180:469-483

College of Veterinary Medicine, University of Missouri, Columbia, MO, USA.

Genetic modifications in combination with highly sophisticated assisted reproductive technologies such as in vitro oocyte maturation and development, in vitro fertilization, intracytoplasmic sperm injection, and in vitro embryo culture have opened many research avenues and treatment options for both animals and humans. The number of genetically modified (GM) rodent strains increased considerably during the last several decades, and their numbers are expected to increase due to efficient gene editing technologies including the CRISPR/Cas9. Rodent ovarian tissues (OT) cryopreservation and transplantation procedures have several applications in biomedical field: they provide a fertility restoration option for GM rodent strains in some circumstances. They also serve as models to investigate OT cryopreservation as potential alternatives for human infertility patients as well as other domestic and wildlife species for the development of improved cryopreservation and subsequent transplantation strategies. The modeling studies enable determining effective cryoprotective agents (CPA), CPA and water permeability kinetics, and cooling and warming rates during the development of OT cryopreservation procedures. Furthermore, rodent models are extremely useful for determining post-thaw OT graft sites as well as potential medical interventions in an effort to expedite angiogenesis and inhibit inflammatory/immune response, OT longevity, and follicular integrity. Here we describe methodologies for rodent OT cryopreservation and potential transplantation sites for frozen-thawed rat and mouse OT.
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http://dx.doi.org/10.1007/978-1-0716-0783-1_22DOI Listing
January 2021

Cryopreservation of Mouse Sperm for Genome Banking.

Methods Mol Biol 2021 ;2180:401-412

College of Veterinary Medicine, University of Missouri, Columbia, MO, USA.

Germplasm cryobanking of transgenic rodent models is a valuable tool for protecting important genotypes from genetic drift, genetic contamination, and loss of breeding colonies due to disease or catastrophic disasters to the housing facilities as well as avoiding stress associated with domestic and international live animal shipment. Furthermore, cryopreservation of germplasm enhances management efficiencies by saving animal room space, reducing workload for staff, reducing cost of maintaining live animals, reducing the number of animals used to maintain a breeding colony, and facilitating transportation of genetics by allowing distribution of frozen germplasm rather than live animals which also reduces the risk of transfer of pathogens between facilities. Thus, effective long-term preservation methods of mouse spermatozoa are critical for future reconstitution of scientifically important mouse strains used for biomedical research.
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http://dx.doi.org/10.1007/978-1-0716-0783-1_17DOI Listing
January 2021

Hypertension and Pathogenic hAPP Independently Induce White Matter Astrocytosis and Cognitive Impairment in the Rat.

Front Aging Neurosci 2020 15;12:82. Epub 2020 Apr 15.

Vulnerable Brain Lab, Department of Anatomy and Cell Biology, Schulich School of Medicine & Dentistry, Western University, London, ON, Canada.

Hypertension is recognized as a risk factor for Alzheimer disease, but the causal link remains undetermined. Although astrocytes and microglia play an important role in maintaining the neurovascular unit, astrocytes and microglia have been understudied in comorbid models of hypertension and Alzheimer disease. In this study, male transgenic Fischer 344 rats (TgAPP21) overexpressing a pathogenic human amyloid precursor protein received 8 weeks of Angiotensin II infusion to increase blood pressure, and the rats were evaluated for astrocytosis, microgliosis, and cognitive function. A linear relationship between astrocytosis and blood pressure was observed in the corpus callosum and cingulum of wildtype rats, with hypertensive wildtype rats matching the elevated baseline astrocytosis seen in normotensive transgenic rats. In contrast, hypertensive transgenic rats did not demonstrate a further increase of astrocytosis, suggesting a deficient response. Angiotensin II infusion did not affect activation of microglia, which were elevated in the white matter and hippocampus of transgenic rats. Angiotensin II infusion did impair both wildtype and transgenic rats' executive functions in the Morris Water Maze. These results present important implications for the interaction between hypertension and pathogenic human amyloid precursor protein expression, as Angiotensin II infusion produced cognitive impairments in both genotypes, but transgenic rats were additionally impaired in developing a normal astrocytic response to elevated blood pressure.
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http://dx.doi.org/10.3389/fnagi.2020.00082DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7174625PMC
April 2020

Morphometric, subcellular, in vitro fertilisation and embryonic developmental assessment of mouse oocytes produced by anti-inhibin serum or pregnant mare serum gonadotrophin superovulation.

Reprod Fertil Dev 2020 Mar;32(5):474-483

Division of Animal Sciences, University of Missouri, 920 East Campus Drive, Columbia, MO 65201, USA; and College of Veterinary Medicine, University of Missouri, 1600 East Rollins Street, Columbia, MO 65211, USA; and Corresponding author. Email:

This study compared the morphometric, subcellular characteristics, in vitro fertilisation (IVF) and embryonic developmental potential of metaphase II (MII) mouse oocytes obtained from females superovulated with either anti-inhibin serum-human chorionic gonadotrophin (AIS-hCG) or pregnant mare serum gonadotrophin (PMSG)-hCG. The oocyte's quantity, quality, zona pellucida (ZP) thickness, perivitelline space (PVS), diameter, microtubules, F-actin, cortical granules (CGs) and mitochondrial distribution were determined. Superovulation using AIS-hCG resulted in a higher numbers of oocyte/donor compared with PMSG-hCG (P=0.002). There was no difference in morphologically normal and abnormal oocytes between AIS-hCG and PMSG-hCG (P=0.425 and P=0.194, respectively). The morphometric measurements showed no difference in oocyte diameter between AIS-hCG and PMSG-hCG (P=0.289). However, the thickness of the ZP of oocytes from AIS-hCG females was decreased compared with PMSG-hCG (P<0.001). The PVS of oocytes from the AIS-hCG was larger than with PMSG-hCG (P<0.001). The microtubules of oocytes from both AIS-hCG and PMSG-hCG were normal, although there was an increased fluorescence intensity in the AIS-hCG oocytes (P<0.001). The F-actin and CGs distribution in oocytes from both AIS-hCG and PMSG-hCG were similar (P=0.330 and P=0.13, respectively). Although the oocytes from PMSG-hCG females had homogenously distributed mitochondria, AIS-hCG oocytes showed more peripheral distribution with no differences in fluorescence intensity (P=0.137). The blastocyst development rates after IVF with fresh sperm showed no difference between AIS-hCG and PMSG-hCG (P=0.235). These data suggested that AIS-hCG superovulation produces high numbers of morphologically normal oocytes that also possess normal subcellular structures, good morphological characteristics and had high invitro embryonic developmental potential.
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http://dx.doi.org/10.1071/RD19131DOI Listing
March 2020

White matter inflammation and cognitive function in a co-morbid metabolic syndrome and prodromal Alzheimer's disease rat model.

J Neuroinflammation 2020 Jan 21;17(1):29. Epub 2020 Jan 21.

Department of Anatomy & Cell Biology, Schulich School of Medicine & Dentistry, Western University, London, ON, N6A 5C1, Canada.

Background: Metabolic syndrome, the development of which is associated with high-caloric Western diet (HCD) intake, represent a risk factor for mild cognitive impairment (MCI) and dementia including Alzheimer's disease (AD) later in life. This study aimed to investigate the effect of diet-induced metabolic disturbances on white matter neuroinflammation and cognitive function in a transgenic (TG) Fischer 344 rat carrying a human β-amyloid precursor protein (APP) gene with Swedish and Indiana mutations (APP21 TG), a model of pre-AD and MCI.

Methods: TG and wildtype (WT) rats received either a HCD with 40% kJ from fat supplemented with 20% corn syrup drink or a standard diet for 12 weeks. Body weight, caloric intake, and blood pressure were measured repeatedly. End-point changes in glucose and lipid metabolism were also assessed. Open field task was used for assessment of activity; Morris water maze was used to assess spatial learning and memory. Cerebral white matter microglia and astrocytes, hippocampal neurons, and neuronal synapses were examined using immunohistochemistry.

Results: Rats maintained on the HCD developed significant obesity, visceral adiposity, dyslipidemia, and hyperinsulinemia, but did not become hypertensive. Impaired glucose tolerance was observed only in WT rats on the HCD. Total microglia number, activated OX-6+ microglia, as well as GFAP+ astrocytes located predominantly in the white matter were greater in the APP21 TG rat model in comparison to WT rats. HCD-driven metabolic perturbations further exacerbated white matter microgliosis and microglia cell activation in the APP21 TG rats and led to detectable changes in spatial reference memory in the comorbid prodromal AD and metabolic syndrome group compared to WT control rats. Neuronal density in the CA1 subregion of the hippocampus was not different between the experimental groups. Synaptic density in the CA1 and CA3 hippocampal subregions was lower in the TG rats compared to WT rats; however, there was no additional effect of the co-morbidity on this measure.

Conclusions: These results suggest that white matter neuroinflammation might be one of the possible processes of early interaction of metabolic syndrome with MCI and pre-AD and could be one of the early brain pathologies contributing to cognitive deficits observed in mild cognitive impairment and dementia, including AD cases.
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http://dx.doi.org/10.1186/s12974-020-1698-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6975033PMC
January 2020

Ovariectomy Influences Cognition and Markers of Alzheimer's Disease.

J Alzheimers Dis 2020 ;73(2):529-541

Department of Veterinary Pathobiology, College of Veterinary Medicine, University of Missouri, Columbia, MO, USA.

Alzheimer's disease (AD) is one of the most devastating and costly diseases, and prevalence of AD increases with age. Furthermore, females are twice as likely to suffer from AD compared to males. The cessation of reproductive steroid hormone production during menopause is hypothesized to cause this difference. Two rodent AD models, APP21 and APP+PS1, and wild type (WT) rats underwent an ovariectomy or sham surgery. Changes in learning and memory, brain histology, amyloid-β (Aβ) deposition, levels of mRNAs involved in Aβ production and clearance, and synaptic and cognitive function were determined. Barnes maze results showed that regardless of ovariectomy status, APP+PS1 rats learned slower and had poor memory retention. Ovariectomy caused learning impairment only in the APP21 rats. High levels of Aβ42 and very low levels of Aβ40 were observed in the brain cortices of APP+PS1 rats indicating limited endogenous PS1. The APP+PS1 rats had 43-fold greater formic acid soluble Aβ42 than Aβ40 at 17 months. Furthermore, levels of formic acid soluble Aβ42 increased 57-fold in ovariectomized APP+PS1 rats between 12 and 17 months of age. The mRNA encoding Grin1 significantly decreased due to ovariectomy whereas levels of Bace1, Chat, and Prkcb all decreased with age. The expression levels of mRNAs involved in Aβ degradation and AβPP cleavage (Neprilysin, Ide, Adam9, and Psenen) were found to be highly correlated with each other as well as hippocampal Aβ deposition. Taken together, these results indicate that both ovariectomy and genotype influence AD markers in a complex manner.
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http://dx.doi.org/10.3233/JAD-190935DOI Listing
November 2020

Euthanasia via CO inhalation causes premature cortical granule exocytosis in mouse oocytes and influences in vitro fertilization and embryo development.

Mol Reprod Dev 2019 07 13;86(7):825-834. Epub 2019 May 13.

Department of Veterinary Pathobiology, College of Veterinary Medicine, University of Missouri, Columbia, Missouri.

Generation of high quality mouse metaphase II oocytes is an integral part for efficient in vitro fertilization (IVF), and subsequent embryo production for reproductive studies and genome banking. The main objectives of this study were to investigate the impact of various euthanasia methods on IVF, embryo development, and subcellular structures of MII mouse oocytes. Following superovulation regimen, female mice were euthanized by high flow CO (H CO ), low flow CO (L CO ), or cervical dislocation (CD). The MII oocytes obtained from these mice were evaluated for subcellular integrity by assessing their cortical granules and F-actin. Furthermore, fertilization and subsequent embryonic development competence up to blastocyst stage were also evaluated in vitro. The oocytes collected from females euthanized by CD resulted in significantly higher two-cell development rates (p = 0.028) and subsequently lead to in higher embryo development rates (p = 0.027) compared with oocytes from females euthanized by L CO . The cortical granule integrity analysis revealed significantly higher rate of premature cortical granules exocytosis (PCGE) for L CO group compared with CD and H CO groups (p < 0.001). These data collectively suggest that CO associated PCGE during euthanasia procedure is the main cause of decreased IVF rates and CD is the optimal euthanasia method for the purpose of obtaining good quality MII oocytes for mouse IVF and other reproductive studies.
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http://dx.doi.org/10.1002/mrd.23167DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7890572PMC
July 2019

Impaired behavioural flexibility related to white matter microgliosis in the TgAPP21 rat model of Alzheimer disease.

Brain Behav Immun 2019 08 15;80:25-34. Epub 2019 Feb 15.

Department of Anatomy & Cell Biology, Schulich School of Medicine & Dentistry, Western University, London, ON, Canada; Department of Clinical Neurological Sciences, University Hospital, Western University, London, ON, Canada. Electronic address:

Executive dysfunction and white matter inflammation continue to be relatively understudied in rodent models of Alzheimer's disease (AD). Behavioural inflexibility is an important component of executive dysfunction that can be further categorized as perseverative or regressive, which respectively specify whether maladaptive persistence occurs early or late during a behavioural change. Previous studies of the TgAPP21 rat model of AD (expressing pathogenic hAPP) suggested a potentially spontaneous increase of regressive behavioral inflexibility. In this study, 7-8-month-old male TgAPP21 rats were tested for behavioral flexibility, learning, and memory using an operant conditioning chamber and the Morris Water Maze (MWM). TgAPP21 rats demonstrated a regressive behavioral inflexibility during set shifting in an operant conditioning chamber (regressive errors η = 0.32 and number of errors after criterion η = 0.33). Regressive behavior was also demonstrated in the MWM probe test, wherein TgAPP21 rats significantly increased their swim time in the target quadrant during the last third of the probe test (43% vs 33% in the first 2 thirds of the probe test or the Wt rats' 29%-32%); this behavioral phenotype has not been previously described in the MWM. TgAPP21 demonstrated further impairment of behavioural inflexibility as they committed a greater number of reversal errors in the operant conditioning chamber (η = 0.30). Diffuse microglia activation was increased in the white matter tracts of TgAPP21 (corpus callosum, cingulum, and internal capsule; η = 0.59-0.62), which was found to correlate with the number of reversal errors in the operant conditioning chamber (R = 0.42). As TgAPP21 rats do not spontaneously develop amyloid plaques but have been shown in previous studies to be vulnerable to the development of plaques, these rats demonstrate an important onset of cognitive change and inflammation in the pre-plaque phase of AD. TgAPP21 rats are also an instrumental model for studying the role and mechanism of white matter microglial activation in executive functioning. This is pertinent to clinical research of prodromal AD which has suggested that white matter inflammation may underlie impairment of executive functions such as behavioral flexibility.
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http://dx.doi.org/10.1016/j.bbi.2019.02.013DOI Listing
August 2019

APP21 transgenic rats develop age-dependent cognitive impairment and microglia accumulation within white matter tracts.

J Neuroinflammation 2018 Aug 28;15(1):241. Epub 2018 Aug 28.

Vulnerable Brain Laboratory, Department of Anatomy and Cell Biology, Schulich School of Medicine and Dentistry, University of Western Ontario, 1151 Richmond St, London, Ontario, N6A 5C1, Canada.

Background: Most of the animal models commonly used for preclinical research into Alzheimer's disease (AD) largely fail to address the pathophysiology, including the impact of known risk factors, of the widely diagnosed sporadic form of the disease. Here, we use a transgenic rat (APP21) that does not develop AD-like pathology spontaneously with age, but does develop pathology following vascular stress. To further the potential of this novel rat model as a much-needed pre-clinical animal model of sporadic AD, we characterize APP21 transgenic rats behaviorally and histologically up to 19 months of age.

Methods: The open field test was used as a measure of activity; and the Morris water maze was used to assess learning, memory, and strategy shift. Neuronal loss and microglia activation were also assessed throughout the brain.

Results: APP21 transgenic rats showed deficits in working memory from an early age, yet memory recall performance after 24 and 72 h was equal to that of wildtype rats and did not deteriorate with age. A deficit in strategy shift was observed at 19 months of age in APP21 transgenic rats compared to Fischer wildtype rats. Histologically, APP21 transgenic rats demonstrated accelerated white matter inflammation compared to wildtype rats, but interestingly no differences in neuron loss were observed.

Conclusions: The combined presence of white matter pathology and executive function deficits mirrored what is often found in patients with mild cognitive impairment or early dementia, and suggests that this rat model will be useful for translationally meaningful studies into the development and prevention of sporadic AD. The presence of widespread white matter inflammation as the only observed pathological correlate for cognitive deficits raises new questions as to the role of neuroinflammation in cognitive decline.
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http://dx.doi.org/10.1186/s12974-018-1273-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6114740PMC
August 2018

Memory deficiency, cerebral amyloid angiopathy, and amyloid-β plaques in APP+PS1 double transgenic rat model of Alzheimer's disease.

PLoS One 2018 11;13(4):e0195469. Epub 2018 Apr 11.

Department of Veterinary Pathobiology, College of Veterinary Medicine, University of Missouri, Columbia, Missouri, United States of America.

Transgenic rat models of Alzheimer's disease were used to examine differences in memory and brain histology. Double transgenic female rats (APP+PS1) over-expressing human amyloid precursor protein (APP) and presenilin 1 (PS1) and single transgenic rats (APP21) over-expressing human APP were compared with wild type Fischer rats (WT). The Barnes maze assessed learning and memory and showed that both APP21 and APP+PS1 rats made significantly more errors than the WT rats during the acquisition phase, signifying slower learning. Additionally, the APP+PS1 rats made significantly more errors following a retention interval, indicating impaired memory compared to both the APP21 and WT rats. Immunohistochemistry using an antibody against amyloid-β (Aβ) showed extensive and mostly diffuse Aβ plaques in the hippocampus and dense plaques that contained tau in the cortex of the brains of the APP+PS1 rats. Furthermore, the APP+PS1 rats also showed vascular changes, including cerebral amyloid angiopathy with extensive Aβ deposits in cortical and leptomeningeal blood vessel walls and venous collagenosis. In addition to the Aβ accumulation observed in arterial, venous, and capillary walls, APP+PS1 rats also displayed enlarged blood vessels and perivascular space. Overall, the brain histopathology and behavioral assessment showed that the APP+PS1 rats demonstrated behavioral characteristics and vascular changes similar to those commonly observed in patients with Alzheimer's disease.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0195469PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5895023PMC
July 2018

Membrane-lipid homeostasis in a prodromal rat model of Alzheimer's disease: Characteristic profiles in ganglioside distributions during aging detected using MALDI imaging mass spectrometry.

Biochim Biophys Acta Gen Subj 2018 06 13;1862(6):1327-1338. Epub 2018 Mar 13.

Department of Anatomy and Cell Biology, Schulich School of Medicine and Dentistry, University of Western Ontario, London, ON, Canada. Electronic address:

Background: Accumulation of simple gangliosides GM2 and GM3, and gangliosides with longer long-chain bases (d20:1) have been linked to toxicity and the pathogenesis of Alzheimer's disease (AD). Conversely, complex gangliosides, such as GM1, have been shown to be neuroprotective. Recent evidence using matrix-assisted laser desorption ionization imaging mass spectrometry (MALDI-IMS) has demonstrated that a-series gangliosides are differentially altered during normal aging, yet it remains unclear how simple species are shifting relative to complex gangliosides in the prodromal stages of AD.

Methods: Ganglioside profiles in wild-type (Wt) and transgenic APP21 Fischer rats were detected and quantified using MALDI-IMS at P0 (birth), 3, 12, and 20 months of age and each species quantified to allow for individual species comparisons.

Results: Tg APP21 rats were found to have a decreased level of complex gangliosides in a number of brain regions as compared to Wt rats and showed higher levels of simple gangliosides. A unique pattern of expression was observed in the white matter as compared to gray matter regions, with an age-dependent decrease in GD1 d18:1 species observed and significantly elevated levels of GM3 in Tg APP21 rats.

Conclusions: These results are indicative of a pathological shift in ganglioside homeostasis during aging that is exacerbated in Tg APP21 rats.

General Significance: Ganglioside dysregulation may occur in the prodromal stages of neurodegenerative diseases like AD.
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http://dx.doi.org/10.1016/j.bbagen.2018.03.011DOI Listing
June 2018

Bone turnover is altered in transgenic rats overexpressing the P2Y2 purinergic receptor.

Purinergic Signal 2017 Dec 21;13(4):545-557. Epub 2017 Aug 21.

College of Veterinary Medicine, University of Missouri, Columbia, MO, USA.

It is now widely recognized that purinergic signaling plays an important role in the regulation of bone remodeling. One receptor subtype, which has been suggested to be involved in this regulation, is the P2Y2 receptor (P2Y2R). In the present study, we investigated the effect of P2Y2R overexpression on bone status and bone cell function using a transgenic rat. Three-month-old female transgenic Sprague Dawley rats overexpressing P2Y2R (P2Y2R-Tg) showed higher bone strength of the femoral neck. Histomorphometry showed increase in resorptive surfaces and reduction in mineralizing surfaces. Both mineral apposition rate and thickness of the endocortical osteoid layer were higher in the P2Y2R-Tg rats. μCT analysis showed reduced trabecular thickness and structural model index in P2Y2R-Tg rats. Femoral length was increased in the P2Y2R-Tg rats compared to Wt rats. In vitro, there was an increased formation of osteoclasts, but no change in total resorption in cultures from P2Y2R-Tg rats. The formation of mineralized nodules was significantly reduced in the osteoblastic cultures from P2Y2R-Tg rats. In conclusion, our study suggests that P2Y2R is involved in regulation of bone turnover, due to the effects on both osteoblasts and osteoclasts and that these effects might be relevant in the regulation of bone growth.
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http://dx.doi.org/10.1007/s11302-017-9582-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5714845PMC
December 2017

Behavioural inflexibility in a comorbid rat model of striatal ischemic injury and mutant hAPP overexpression.

Behav Brain Res 2017 08 8;333:267-275. Epub 2017 Jul 8.

Department of Anatomy & Cell Biology, Schulich School of Medicine & Dentistry, Western University, London ON, Canada. Electronic address:

Alzheimer disease (AD) and stroke coexist and interact; yet how they interact is not sufficiently understood. Both AD and basal ganglia stroke can impair behavioural flexibility, which can be reliably modeled in rats using an established operant based set-shifting test. Transgenic Fischer 344-APP21 rats (TgF344) overexpress pathogenic human amyloid precursor protein (hAPP) but do not spontaneously develop overt pathology, hence TgF344 rats can be used to model the effect of vascular injury in the prodromal stages of Alzheimer disease. We demonstrate that the injection of endothelin-1 (ET1) into the dorsal striatum of TgF344 rats (Tg-ET1) produced an exacerbation of behavioural inflexibility with a behavioural phenotype that was distinct from saline-injected wildtype & TgF344 rats as well as ET1-injected wildtype rats (Wt-ET1). In addition to profiling the types of errors made, interpolative modeling using logistic exposure-response regression provided an informative analysis of the timing and efficiency of behavioural flexibility. During set-shifting, Tg-ET1 committed fewer perseverative errors than Wt-ET1. However, Tg-ET1 committed significantly more regressive errors and had a less efficient strategy change than all other groups. Thus, behavioural flexibility was more vulnerable to striatal ischemic injury in TgF344 rats.
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http://dx.doi.org/10.1016/j.bbr.2017.07.006DOI Listing
August 2017

Direct Evidence for P2Y2 Receptor Involvement in Vascular Response to Injury.

J Vasc Res 2016 11;53(3-4):163-171. Epub 2016 Oct 11.

Department of Veterinary Pathobiology, College of Veterinary Medicine, University of Missouri, Columbia, Mont., USA.

Objectives: Extracellular nucleotide release at the site of arterial injury mediates the proliferation and migration of vascular smooth muscle cells. Our aim was to investigate the role of the P2Y2 nucleotide receptor (P2Y2R) in neointimal hyperplasia. Approach and Results: Vascular injury was induced by the implantation of a polyethylene cuff around the femoral artery in wild-type and P2Y2R-deficient mice (P2Y2R-/-). Electron microscopy was used to analyze monocyte and lymphocyte influx to the intima 36 h after injury. Compared to wild-type littermates, P2Y2R-/- mice exhibited a 3-fold decreased number of mononuclear leukocytes invading the intima (p < 0.05). Concomitantly, the migration of smooth muscle cells was decreased by more than 60% (p < 0.05), resulting in a sharp inhibition of intimal thickening formation in P2Y2R-/- mice (n = 15) 14 days after cuff placement. In vitro, loss of P2Y2R significantly impaired monocyte migration in response to nucleotide agonists. Furthermore, transgenic rats overexpressing the P2Y2R developed accelerated intimal lesions resulting in more than 95% luminal stenosis (p < 0.05, n = 10).

Conclusions: Loss- and gain-of-function approaches established direct evidence for P2Y2R involvement in neointimal hyperplasia. Specific anti-P2Y2R therapies may be used against restenosis and bypass graft failure.
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http://dx.doi.org/10.1159/000449059DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5154871PMC
May 2017

Presenilin 1 transgene addition to amyloid precursor protein overexpressing transgenic rats increases amyloid beta 42 levels and results in loss of memory retention.

BMC Neurosci 2016 07 7;17(1):46. Epub 2016 Jul 7.

Department of Veterinary Pathobiology, College of Veterinary Medicine, University of Missouri, 1600 East Rollins Street, Room W191, Columbia, MO, 65211, USA.

Background: We previously reported the production of transgenic rats (APP21 line) that over-express human amyloid precursor protein (APP) containing Swedish and Indiana mutations. In order to generate a better model for Alzheimer's disease (AD), the APP21 rat line was used to generate double transgenic line that over-expressed Presenilin 1 (PS1) with L166P mutation in addition to APP transgene (APP + PS1 line).

Results: Thirty-two double transgenic founders were generated and the ultimate transgenic founder was selected based on PS1 transgene copy number and level of amyloid-beta (Aβ)42 peptide. The APP + PS1 double transgenic rats had 38 times more PS1 in brains compared to APP rats. Behavioral assessment using Barnes maze showed that APP + PS1 rats exhibited a larger learning and memory deficit than APP21 rats. Double transgenic rats also produced more Aβ42. Histological examination of the brains showed that the APP21 rat line displayed neurofibrillary tangles and in contrast, the APP + PS1 line showed chromatolysis in hippocampal neurons and neuronal loss in CA3 region of hippocampus.

Conclusions: Due to the separate segregation of APP and PS1 transgenes in APP + PS1 double transgenic rats, this transgenic line may be a valuable model for studying the effects of various levels of APP and PS1 transgenes on various aspects of brain pathologies associated with the AD phenotype.
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http://dx.doi.org/10.1186/s12868-016-0281-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4936262PMC
July 2016

Post-thaw ATP supplementation enhances cryoprotective effect of iodixanol in rat spermatozoa.

Reprod Biol Endocrinol 2016 Jan 29;14. Epub 2016 Jan 29.

Department of Veterinary Pathobiology, College of Veterinary Medicine, University of Missouri-Columbia, 1600 East Rollins Street, Room W191, Columbia, MO, 65211, USA.

Background: Successful cryopreservation of rat spermatozoa from various strains still remains a challenge. The objective of this study was to determine if combinations of OptiPrep™ (iodixanol) and adenosine 5'-triphosphate (ATP) can improve rat sperm function during the cryopreservation procedure.

Methods: Epididymal rat spermatozoa were frozen under different OptiPrep™ concentrations (0, 1, 2, 3 or 4 %) and were diluted with media supplemented with or without 2 mM ATP after thawing. Post-thaw sperm motility, acrosomal membrane integrity (AMI) and mitochondrial membrane potential (MMP) were then evaluated. In addition, the effect of different OptiPrep™ concentrations on fresh and cooled rat spermatozoa was tested via motility.

Results: There was no effect of OptiPrep™ on motility of fresh and cooled spermatozoa. The supplementation of 1 and 2 % OptiPrep™ increased motility of frozen spermatozoa at 10 min after thawing, while it did not improve motility of spermatozoa at 3 h after thawing in the absence of ATP. During incubation of thawed spermatozoa, the ATP addition protected time-dependent decrease in motility after thawing in OptiPrep™-treated samples. OptiPrep™ had no effect on AMI and MMP in frozen-thawed spermatozoa but combinations of OptiPrep™ and ATP improved MMP in frozen-thawed spermatozoa.

Conclusions: Iodixanol has cryoprotective effects during rat sperm freezing without any toxic effect. Moreover, the combinations of iodixanol and ATP have a beneficial role in maintaining function of frozen-thawed rat spermatozoa for long period of incubation post-thaw.
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http://dx.doi.org/10.1186/s12958-016-0141-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4731941PMC
January 2016

Kaolin-induced chronic hydrocephalus accelerates amyloid deposition and vascular disease in transgenic rats expressing high levels of human APP.

Fluids Barriers CNS 2015 24;12(1). Epub 2015 Jan 24.

Department of Neurosurgery, The Warren Alpert Medical School of Brown University and the Aldrich Laboratories at Rhode Island Hospital, 593 Eddy Street, Providence, RI 02903 USA ; Department of Pathology (Neuropathology), Warren Alpert Medical School of Brown University and the Aldrich Laboratories at Rhode Island Hospital, 593 Eddy Street, Providence, RI 02903 USA.

Background: Normal pressure hydrocephalus (NPH) is most common in the elderly and has a high co-morbidity with Alzheimer's disease (AD) and cerebrovascular disease (CVD). To understand the relationship between NPH, AD and CVD, we investigated how chronic hydrocephalus impacts brain amyloid-beta peptide (Aβ) accumulation and vascular pathology in an AD transgenic rodent model. Previously we showed that the altered CSF physiology produced by kaolin-hydrocephalus in older wild-type Sprague-Dawley rats increased Aβ and hyperphosphorylated Tau (Silverberg et. al. Brain Res. 2010, 1317:286-296). We postulated that hydrocephalus would similarly affect an AD rat model.

Methods: Thirty-five transgenic rats (tgAPP21) that express high levels of human APP and naturally overproduce Aβ40 were used. Six- (n = 7) and twelve-month-old (n = 9) rats had hydrocephalus induced by cisternal kaolin injection. We analyzed Aβ burden (Aβ40, Aβ42 and oligomeric Aβ) and vascular integrity (Masson trichrome and Verhoeff-Van Gieson) by immunohistochemistry and chemical staining at 10 weeks (n = 8) and 6 months (n = 5) post hydrocephalus induction. We also analyzed whether the vascular pathology seen in tgAPP21 rats, which develop amyloid angiopathy, was accelerated by hydrocephalus. Age-matched naïve and sham-operated tgAPP21 rats served as controls (n = 19).

Results: In hydrocephalic tgAPP21 rats, compared to naïve and sham-operated controls, there was increased Aβ 40 and oligomeric Aβ in hippocampal and cortical neurons at 10 weeks and 6 months post-hydrocephalus induction. No dense-core amyloid plaques were seen, but diffuse Aβ immunoreactivity was evident in neurons. Vascular pathology was accelerated by the induction of hydrocephalus compared to controls. In the six-month-old rats, subtle degenerative changes were noted in vessel walls at 10 weeks post-kaolin, whereas at six months post-kaolin and in the 12-month-old hydrocephalic rats more pronounced amyloid angiopathic changes were seen, with frequent large areas of infarction noted.

Conclusions: Kaolin-hydrocephalus can accelerate intraneuronal Aβ40 accumulation and vascular pathology in tgAPP21 rats. In addition, disrupted CSF production and reduced CSF turnover results in impaired Aβ clearance and accelerated vascular pathology in chronic hydrocephalus. The high co-morbidity seen in NPH, AD and CVD is likely not to be an age-related coincidence, but rather a convergence of pathologies related to diminished CSF clearance.
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http://dx.doi.org/10.1186/2045-8118-12-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4328504PMC
February 2015

Molecular and ultrastuctural changes of rat pre-implantation embryos during two-cell developmental arrest.

J Assist Reprod Genet 2014 Jun;31(6):767-80

Background: Rat pre-implantation embryos often suffer 2-cell stage developmental arrest and fail to progress further under in-vitro conditions.

Objective: In order to understand underlying mechanism leading to 2-cell arrest, we investigated the molecular changes, culture conditions and subcellular changes.

Methods: Gene expression in in-vivo developed 2-cell embryos (in-vivo), in- vitro developed 2-cell embryos (in-vitro), and in-vitro 2-cell arrested embryos (arrested) were investigated using microarrays and real-time PCR. Ultra-structural changes were determined using electron microscopy.

Results: Gene expression was similar between in-vivo and in-vitro embryos. Over 2400 genes changed in arrested embryos compared to in-vivo and in-vitro embryos. The mRNAs encoding proteins involved in translation were elevated in arrested embryos. In-vivo and in-vitro embryos highly expressed genes that were involved in cell cycle, and protein catabolic process compared to arrested embryos. Gene expression data suggested subcellular changes associated with 2-cell block. Transmission electron microscopy showed that in-vivo embryos had healthy subcellular structure, whereas arrested embryos did not have a nuclear membrane, contained small mitochondria and autophagic vacuoles. Furthermore, gene expression data was used for the optimization of culture media conditions to obtain better in-vitro embryonic development. Comparison of five and 20 % oxygen in culture resulted in two times more blastocyst formation with 5 % oxygen.

Conclusions: These results showed that although all experimental groups appeared morphologically similar, arrested embryos had ultra-structural and molecular changes associated with oxidative stress and apoptosis. In-vitro culture under low oxygen and media additives reduced 2-cell block in rat embryos.
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http://dx.doi.org/10.1007/s10815-014-0213-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4048373PMC
June 2014

Short-term storage of rat sperm in the presence of various extenders.

J Am Assoc Lab Anim Sci 2013 Nov;52(6):732-7

Department of Veterinary Pathobiology, College of Veterinary Medicine, University of Missouri, Columbia, Missouri, USA.

Sperm preservation protocols differ among animal species because of different sperm characteristics among species. Rat sperm have extreme sensitivity to suboptimal conditions in centrifugation, pipetting and chilling due to their longer tail, the shape and size of the sperm head, and membrane composition. The aim of this study was to determine optimal conditions for short-term storage of rat sperm by evaluating their motility and membrane and acrosomal integrity in response to various extender solutions, temperatures, and durations. Motility of rat sperm was highest when stored at 22 °C; motility was 28% and 14% at 72 h in TL-HEPES and PBS extenders, respectively. The motility and membrane integrity of rat sperm fell significantly within 24 h at 4 and 37 °C. Although cold storage did not have a detrimental effect on acrosomal integrity of sperm, room temperature storage reduced acrosomal integrity after 24 h. LEY extender caused the highest loss in acrosomal integrity at 48 and 72 h. In conclusion, storage at 4 or 37 ° C reduced the motility and membrane integrity of rat sperm even with short incubation periods. Rat sperm stored in TL-HEPES or PBS remained motile for at least 3 d when held at 22 °C.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3838607PMC
November 2013

The effects of cooling rates and type of freezing extenders on cryosurvival of rat sperm.

Cryobiology 2013 Oct 30;67(2):109-16. Epub 2013 May 30.

University of Missouri, College of Veterinary Medicine, Dept. of Veterinary Pathobiology, 1600 East Rollins Street, Columbia, MO, 65211 USA.

Cryopreservation of rat sperm is very challenging due to its sensitivity to various stress factors. The objective of this study was to determine the optimal cooling rate and extender for epididymal sperm of outbred Sprague Dawley (SD) and inbred Fischer 344 (F344) rat strains. The epididymal sperm from 10 to 12 weeks old sexually mature SD and F344 strains were suspended in five different freezing extenders, namely HEPES buffered Tyrode's lactate (TL-HEPES), modified Kreb's Ringer bicarbonate (mKRB), 3% dehydrated skim milk (SM), Salamon's Tris-citrate (TRIS), and tes/tris (TES). All extenders contained 20% egg yolk, 0.75% Equex Paste and 0.1 M raffinose or 0.1 M sucrose. The sperm samples in each extender were cooled to 4°C and held for 45 min for equilibration before freezing. The equilibrated sperm samples in each extender were placed onto a shallow quartz dish inserted into Linkam Cryostage (BCS 196). The samples were then cooled to a final temperature of -150°C by using various cooling rates (10, 40, 70, and 100°C/min). For thawing, the quartz dish containing the sperm samples were rapidly removed from the Linkam cryo-stage and placed on a 37°C slide warmer and held for 1 min before motility analysis. Sperm membrane and acrosomal integrity and mitochondrial membrane potential (MMP) were assessed by SYBR-14/Propidium iodide, Alexa Fluor-488-PNA conjugate and JC-1, respectively. The total motility, acrosomal integrity, membrane integrity and MMP values were compared among cooling rates and extenders. Both cooling rate and type of extender had significant effect on cryosurvival (P < 0.05). Sperm motility increased as cooling rate was increased for both strains (P < 0.05). Highest cryosurvival was achieved when 100°C/min cooling rate was used in combination with TES extender containing 20% egg yolk, 0.75% Equex paste and either 0.1M sucrose or raffinose (P < 0.05). This study showed that TES extender containing 0.1 M raffinose or sucrose with 70°C/min and 100°C/min cooling rate improved post-thaw motility of rat sperm.
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http://dx.doi.org/10.1016/j.cryobiol.2013.05.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3772956PMC
October 2013

Comparison of cryoprotective effects of iodixanol, trehalose and cysteamine on ram semen.

Anim Reprod Sci 2013 Jun 30;139(1-4):38-44. Epub 2013 Mar 30.

Department of Reproduction and Artificial Insemination, Faculty of Veterinary Medicine, Dicle University, 21280, Diyarbakir, Turkey.

This study was conducted to improve cryosurvival of electroejaculated (EE) ram semen in the presence of iodixanol (OptiPrep™), trehalose or cysteamine. A tris-based extender was used to prepare 12 extenders containing OptiPrep™ (Op), trehalose (Tr) or cysteamine (Cy) alone, or different combinations of these compounds. Extenders were designated as follows: Tris (control), Op1.25 (1.25% Op, v/v), Op2.5 (2.5% Op, v/v), Op5 (5% Op, v/v), Tr50 (50mM Tr), Tr100 (100mM Tr), Cy (5mM Cy), OpTr (2.5% Op and 100mM Tr), OpCy (2.5% Op and 5mM Cy), TrCy (100mM Tr and 5mM Cy), OpTrCy1 (2.5% Op, 100mM Tr and 5mM Cy) and OpTrCy2 (1.25% Op, 50mM Tr and 2.5mM Cy). A two-step dilution was used and glycerol was added at 5°C in the second step. Diluted samples were equilibrated for 1h, loaded in 0.25mL straws and frozen in a programmable freezing machine. Supplementation of 5% OptiPrep™ significantly protected post-thaw progressive motility, membrane integrity, acrosomal integrity and morphological damages. Trehalose supplementation protected membrane integrity of ram sperm; however, it did not help post-thaw motility and morphology. Supplementation of 5mM cysteamine had detrimental effect on cryosurvival of EE ram semen. These results demonstrate that the supplementation of iodixanol increases the cryosurvival of EE ram semen in a dose-dependent manner.
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http://dx.doi.org/10.1016/j.anireprosci.2013.03.010DOI Listing
June 2013

Estrus synchronization and ovarian hyper-stimulation treatments have negligible effects on cumulus oocyte complex gene expression whereas induction of ovulation causes major expression changes.

Mol Reprod Dev 2013 Feb;80(2):102-17

Department of Veterinary Pathobiology, College of Veterinary Medicine, University of Missouri-Columbia, Columbia, Missouri 65211, USA.

The effects of exogenous hormones, used for estrus synchronization and ovarian hyper stimulation, on cumulus oocyte complexes (COCs) gene expression in sexually mature rats were determined using microarrays. Gene expression in COCs collected from GnRH (G(trt)), GnRH + eCG (G + E(trt)), and GnRH + eCG + hCG (G + E + H(trt)) treatments were compared to COCs from naturally cycling (NC) rats before the preovulatory luteninizing hormone surge. There was no significant difference in gene expression among NC, G(trt), and G + E(trt); however, over 2,600 genes were significantly different between NC and G + E + H(trt) (P < 0.05). Genes upregulated in G + E + H(trt) encode for: proteins that are involved in prostaglandin synthesis (Ptgs2, Pla2g4a, and Runx1) and cholesterol biosynthesis (Hmgcr, Sc4mol, and Dhcr24); receptors that allow cholesterol uptake (Ldlr and Scarb1), regulate progesterone synthesis (Star), and inactivate estrogen (Sult1e1); and downstream effectors of LH signal (Pgr, Cebpb, Creb3l1, Areg, Ereg, and Adamts1). Conversely, G + E + H(trt) downregulated genes encoding proteins involved in: DNA replication and cell cycle progression (Ccne2, Orc5l, Rad50, and Mcm6); reproductive developmental process; and granulosa cell expansion (Gdf9, Bmp15, Amh, Amhr2, Bmpr1b, Tgfb2, Foxl2, Pde3a, Esr2, Fshr, Ybx2, Ccnd2, Ccnb1ip1, and Zp3); maternal effect genes required for embryo development (Zar1, Npm2, Nlrp5, Dnmt1, H1foo, and Zfp57); amino acid degradation; and ketogenesis (Hmgcs2, and Cpt1b). These results from the rat show that hormones used for estrus synchronization (G(trt)) and ovarian hyper stimulation (G + E(trt)) had minimal effects on gene expression, whereas induction of ovulation (G + E + H(trt)) caused major changes in gene expression of rat COCs. This study provides comprehensive information about regulated genes during late follicle development and ovulation induction.
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http://dx.doi.org/10.1002/mrd.22141DOI Listing
February 2013

Effects of various physical stress factors on mitochondrial function and reactive oxygen species in rat spermatozoa.

Reprod Fertil Dev 2013 ;25(7):1051-64

Department of Veterinary Pathobiology, College of Veterinary Medicine, University of Missouri-Columbia, Columbia, Missouri 65211, USA.

The aim of the present study was to evaluate the effects of various physical interventions on the function of epididymal rat spermatozoa and determine whether there are correlations among these functional parameters. Epididymal rat spermatozoa were subjected to various mechanical (pipetting, centrifugation and Percoll gradient separation) and anisotonic conditions, and sperm motility, plasma membrane integrity (PMI), mitochondrial membrane potential (MMP) and intracellular reactive oxygen species (ROS) were evaluated. Repeated pipetting caused a loss in motility, PMI and MMP (P<0.05). Minimal centrifugation force (200 g) had no effect on motility, PMI and MMP, whereas an increase in the centrifugation force to 400 g or 600 g decreased sperm function (P<0.005). Percoll gradient separation increased total motility, PMI and MMP (P<0.05). However, the spermatozoa that were subjected to mechanical interventions showed high susceptibility to a ROS stimulant (P<0.005). Anisotonic conditions decreased motility, PMI and MMP, and hypotonic conditions in particular increased basal ROS (P<0.05). In correlation tests, there were strong positive correlations among total motility, PMI and MMP, whereas ROS showed no or negatively weak correlations with the other parameters. In conclusion, the physical interventions may act as important variables, affecting functional parameters of epididymal rat spermatozoa. Therefore, careful consideration and proper protocols for handling of rat spermatozoa and osmotic conditions are required to achieve reliable results and minimise damage.
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http://dx.doi.org/10.1071/RD12212DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3706545PMC
March 2014

Gene expression profiles of vitrified in vitro- and in vivo-derived bovine blastocysts.

Mol Reprod Dev 2012 Sep 26;79(9):613-25. Epub 2012 Jul 26.

TUBITAK MRC Genetic Engineering and Biotechnology Institute, Gebze, Turkey.

Vitrification is becoming a preferred method for pre-implantation embryo cryopreservation. The objective of this study was to determine the differentially expressed genes of in vivo- and in vitro-produced bovine embryos after vitrification. In vitro- (IVF) and in vivo-derived (IVV) bovine blastocysts were identified as follows: in vitro-produced fresh (IVF-F), in vitro-produced vitrified (IVF-V), in vivo-derived fresh (IVV-F), in vivo-derived vitrified (IVV-V). The microarray results showed that 53 genes were differentially regulated between IVF and IVV, and 121 genes were differentially regulated between fresh and vitrified blastocysts (P < 0.05). There were 6, 268, 962, and 17 differentially regulated genes between IVF-F × IVV-F, IVF-V × IVV-V, IVF-F × IVF-V, and IVV-F × IVV-V, respectively (P < 0.05). While gene expression was significantly different between fresh and vitrified IVF blastocysts (P < 0.05), it was similar between fresh and vitrified IVV blastocysts. Significantly up-regulated KEGG pathways included ribosome, oxidative phosphorylation, spliceosome, and oocyte meiosis in the fresh IVF blastocyst samples, while sphingolipid and purine metabolisms were up-regulated in the vitrified IVF blastocyst. The results showed that in vitro bovine blastocyst production protocols used in this study caused no major gene expression differences compared to those of in vivo-produced blastocysts. After vitrification, however, in vitro-produced blastocysts showed major gene expression differences compared to in vivo blastocysts. This study suggests that in vitro-produced embryos are of comparable quality to their in vivo counterparts. Vitrification of in vitro blastocysts, on the other hand, causes significant up-regulation of genes that are involved in stress responses.
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http://dx.doi.org/10.1002/mrd.22068DOI Listing
September 2012

Changes in rat spermatozoa function after cooling, cryopreservation and centrifugation processes.

Cryobiology 2012 Dec 30;65(3):215-23. Epub 2012 Jun 30.

Department of Veterinary Pathobiology, College of Veterinary Medicine, University of Missouri-Columbia, Columbia, MO 65211, USA.

Rat sperm cryopreservation is an effective method of archiving valuable strains for biomedical research and handling of rat spermatozoa is very important for successful cryopreservation. The aim of this study was to evaluate changes in rat sperm function during cryopreservation and centrifugation. Epididymal rat spermatozoa were subjected to cooling and freezing-thawing processes and then motility, plasma membrane integrity (PMI), mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) were compared before and after minimum centrifugation force (200×g). Cryopreservation decreased sperm motility, PMI, and MMP (P<0.05). Basal (without ROS inducer, tert-butyl hydroperoxide [TBHP] treatment) and stimulated ROS (with TBHP treatment) were increased in viable cooled spermatozoa compared to viable fresh spermatozoa (P<0.01), with equal susceptibility to TBHP among fresh, cooled, and frozen-thawed spermatozoa. Centrifugation decreased motility and PMI of frozen-thawed spermatozoa (P<0.05). Centrifugation decreased basal ROS of all spermatozoa (P<0.01), while it led to higher susceptibility to TBHP in viable cooled spermatozoa, showing higher increased fold in ROS and decreased rate in viability by TBHP in viable cooled spermatozoa (P<0.05). Cooling process was the major step of ROS generation, with loss in sperm motility, PMI, and MMP. Centrifugation affected function of cryopreserved spermatozoa. These data suggest that centrifugation makes rat spermatozoa susceptible to external ROS source, in particular during cooling process. Thus, protection from ROS damage and minimizing centrifugation should be considered during cryopreservation and post-thaw use of cryopreserved epididymal rat spermatozoa.
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http://dx.doi.org/10.1016/j.cryobiol.2012.06.006DOI Listing
December 2012

P2Y2 receptor-mediated lymphotoxin-α secretion regulates intercellular cell adhesion molecule-1 expression in vascular smooth muscle cells.

J Biol Chem 2012 Mar 1;287(13):10535-10543. Epub 2012 Feb 1.

Departments of Immunology and Microbiology, Indiana University School of Medicine, Indianapolis, Indiana 46202 and.

The proinflammatory cytokine lymphotoxin-α (LTA) is thought to contribute to the pathogenesis of atherosclerosis. However, the mechanisms that regulate its expression in vascular smooth muscle cells (VSMC) are poorly understood. The ability of exogenous nucleotides to stimulate LTA production was evaluated in VSMC by ELISA. The P2Y(2) nucleotide receptor (P2Y(2)R) agonist UTP stimulates a strong and sustained release of LTA from WT but not P2Y(2)R(-/-) SMC. Assessment of LTA gene transcription by LTA promoter-luciferase construct indicated that LTA levels are controlled at the level of transcription. We show using RNAi techniques that knockdown of the actin-binding protein filamin-A (FLNa) severely impaired nucleotide-induced Rho activation and consequent Rho-mediated LTA secretion. Reintroduction of FLNa in FLNa RNAi SMC rescued UTP-induced LTA expression. In addition, we found that UTP-stimulated LTA secretion is not sensitive to brefeldin A, which blocks the formation of vesicles involved in protein transport from the endoplasmic reticulum to the Golgi apparatus, suggesting that P2Y(2)R/filamin-mediated secretion of LTA is independent of the endoplasmic reticulum/Golgi secretory vesicle route. Furthermore, UTP selectively induces ICAM-1 expression in WT but not SMC expressing a truncated P2Y(2)R deficient in LTA secretion. These data suggest that P2Y(2)R recruits FLNa to provide a cytoskeletal scaffold necessary for Rho signaling pathway upstream of LTA release and subsequent stimulation of ICAM-1 expression on vascular smooth muscle cells.
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http://dx.doi.org/10.1074/jbc.M111.313189DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3323005PMC
March 2012

Effect of chilling on the motility and acrosomal integrity of rat sperm in the presence of various extenders.

J Am Assoc Lab Anim Sci 2009 Sep;48(5):499-505

Department of Animal Reproduction and Artificial Insemination, University of Ankara, Ankara, Turkey.

Studies were conducted to determine the effect of chilling on rat sperm and optimal components (extenders) to avoid chilling-induced injury. In the first experiment, the effects of chilling (at 4, 10, or 22 degrees C) on the motility and acrosomal integrity of epididymal sperm from 2 strains of rats (Sprague-Dawley and Fischer 344, F344) were compared. In the second experiment, the motility of epididymal Sprague-Dawley rat sperm after exposure to extenders (HEPES-buffered Tyrode lactate, skim milk, lactose monohydrate, Tris-citrate, and TEST) and cooling and warming was determined. We tested the effects of supplementing base extender solutions with 20% lactose-egg yolk (LEY) alone or in combination with a commercial SDS-based paste (0.5%, v/v) in preventing chilling injury. The motility after each treatment was determined after both cooling and warming. In the third experiment, the motility of Sprague-Dawley rat sperm were compared after supplementing the base extenders with either 0.4 M permeating cryoprotective agent (CPA; glycerol, ethylene glycol, propylene glycol, or DMSO) or 0.1 M nonpermeating CPA (raffinose and sucrose) after cooling and warming. The results showed that chilling significantly reduced the motility-but not acrosomal integrity-of Sprague-Dawley and F344 sperm. Neither motility nor acrosomal integrity differed between Sprague-Dawley and F344 strains. The addition of LEY into each extender significantly prevented motility loss after chilling. These results will be useful during the preparation of optimal extenders and development of successful cryopreservation protocol for rat sperm.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2755019PMC
September 2009

Effects of nonylphenol on motility and subcellular elements of epididymal rat sperm.

Reprod Toxicol 2009 Dec 16;28(4):542-9. Epub 2009 Jun 16.

Afyonkarahisar Kocatepe University, Department of Medical Biology and Genetics, College of Veterinary Medicine, Afyonkarahisar, 03200 Turkey.

Nonylphenol (NP) is an important environmental toxicant and potential endocrine disrupting chemical. The objective of these studies was to determine the effects of NP on epididymal rat sperm in vitro. Epididymal sperm samples from Sprague-Dawley rats were incubated in 1, 10, 100, 250, and 500 microg/ml NP for 1, 2, 3, or 4h. Computer-assisted sperm analysis was used to determine motility. Epifluorescent microscopy was used to determine acrosomal status and flow cytometry was used to determine mitochondrial membrane potential (MMP) and chromatin integrity. Exposure of epididymal rat sperm to 250 or 500 microg/ml NP was highly detrimental to motility (P<0.05), with complete loss of motility observed after exposure to 500 microg/ml NP (P<0.05). The acrosomal integrity of sperm was significantly reduced with the lowest concentration (1 microg/ml) of NP, and higher concentrations resulted in a dose-dependent induction of the acrosomal reaction (P<0.05). Similarly, the percentage of sperm with high MMP declined dramatically after exposure to 100, 250, and 500 microg/ml NP (P<0.05). Duration of NP exposure did not have any effect on motility or MMP and NP did not appear to have detrimental effects on chromatin integrity (P>0.05). These results indicate that major mechanism of action of NP on rat sperm is by adversely affecting their acrosomal integrity. However, NP-induced impaired sperm motility, decreased mitochondrial membrane potential also likely to play an important role in destruction of sperm function.
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http://dx.doi.org/10.1016/j.reprotox.2009.06.007DOI Listing
December 2009

Gene expression profile of rat ovarian tissue following xenotransplantation into immune-deficient mice.

Reproduction 2009 Jun 12;137(6):957-67. Epub 2009 Mar 12.

Department of Veterinary Pathobiology, College of Veterinary Medicine, University of Missouri, Columbia, Missouri 65211, USA.

Immune-compromised mice have been used as gonadal tissue recipients to develop gametes of various mammalian species. The aim of this research was to determine gene expression differences between fresh and frozen-thawed rat xenotransplanted (XT) ovaries as well the gene expression differences between XT and sexually mature rat ovaries that were non-transplanted (NT). Ovaries from sexually immature female rats were transplanted under the kidney capsule of ovariectomized athymic nude mice either fresh or after freezing. The XT ovaries were collected approximately 10-12 weeks after xenografting for microarray analysis. The NT ovaries were collected from sexually mature rats. Gene expression was very similar between fresh and cryopreserved XT ovaries: 125 genes were twofold up- or downregulated, but level of regulation was not statistically significant. Overall patterns of gene expression between XT and NT ovaries were very different indicated by the absence of diagonal relationship between XT and NT ovary gene expression. More than 3000 genes were significantly (P<0.01) up- or downregulated between XT and NT ovaries. Genes involved in metabolic processes, lipid metabolism, and growth were downregulated in XT ovaries, whereas genes involved in immune and inflammatory response were upregulated in XT ovaries. The results showed that ovarian tissue xenografting significantly alters genes responsible for ovarian metabolism and function and leads to an upregulation of genes responsible for graft rejection.
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http://dx.doi.org/10.1530/REP-09-0048DOI Listing
June 2009

Development of a novel transgenic rat overexpressing the P2Y(2) nucleotide receptor using a lentiviral vector.

J Vasc Res 2009 21;46(5):447-58. Epub 2009 Jan 21.

Department of Veterinary Pathobiology, College of Veterinary Medicine, University of Missouri, Columbia, Mo. 65211, USA.

The G protein-coupled P2Y(2) nucleotide receptor (P2Y(2)R) is upregulated in response to stress and tissue injury and has been postulated to play a role in chronic inflammation seen in atherosclerosis, Alzheimer's disease and Sjogren's syndrome. The role of P2Y(2)R upregulation in vivo is poorly understood, in part due to the lack of a P2Y(2)R overexpressing animal model. The P2Y(2)R overexpressing transgenic rat was generated using a lentiviral vector. Rats overexpressing P2Y(2)R showed a significant increase in P2Y(2)R mRNA levels in all tissues screened as compared to nontransgenic rats. Fura 2 imaging of smooth muscle cells (SMCs) isolated from aorta indicated that the percentage of cells exhibiting increases in the intracellular free calcium concentration in response to P2Y(2)R agonists was significantly greater in freshly isolated SMCs from transgenic rats than wild-type controls. Histopathological examination of tissues revealed that P2Y(2)R overexpressing rats develop lymphocytic infiltration in lacrimal glands and kidneys as early as at 3 months of age. These rats show similarities to patients with Sjogren's syndrome who display lymphocyte-mediated tissue damage. This transgenic rat model of P2Y(2)R overexpression may prove useful for linking P2Y(2)R upregulation with chronic inflammatory diseases, neurodegenerative diseases and Sjogren's syndrome.
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http://dx.doi.org/10.1159/000194274DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3593343PMC
September 2009