Yuksel Agca

Osmotic Characteristics of Mouse Spermatozoa in the Presence of Extenders and Sugars

Abstract Successful cryopreservation requires cells to tolerate volume excursions experienced during permeating cryoprotectant equilibration and during cooling and warming. However, prior studies have demonstrated that mouse spermatozoa are extremely sensitive to osmotically induced volume changes. A series of three experiments were conducted 1) to test the efficacy of two commonly used extender media components, egg yolk (EY) and skim milk (SM), in broadening the osmotic tolerance limits (OTL) of ICR and B6C3F1 murine spermatozoa; 2) to determine if the extender components affected sperm plasma membrane permeability coefficients for water and cryoprotective agent (CPA) characteristics; and 3) to test the effects of permeating and nonpermeating CPA on mouse sperm morphology. In experiment 1, sperm samples were added to 150, 225, 300, 450, or 600 mOsm NaCl, EY, SM, sucrose, or choline chloride at 22°C and then returned to isosmotic conditions. In experiment 2, epididymal sperm were preequilibrated in 1 M glycerol (Gly) or 2 M ethylene glycol (EG) prepared in SM extender, abruptly exposed to isosmotic conditions at 22, 15, or 2°C, and the corresponding volume excursions were measured and analyzed. In experiment 3, the effects of permeating CPA (0.3 M EG or dimethyl sulfoxide) or nonpermeating CPA (12% sucrose or 18% raffinose) on sperm morphology (i.e., principle midpiece folding and putative membrane fusion) were evaluated. Experiment 1 showed that spermatozoa from ICR and B6C3F1 mice have effectively broader OTL when exposed to EY or SM extenders. The results of experiment 2 indicated that, for ICR sperm, the activation energy (Ea) for the hydraulic conductivity (Lp) was unchanged in SM extender. However, for B6C3F1 sperm, there were significant differences in Ea of Lp in the presence of Gly and EG. The result of experiment 3 indicated that permeating CPAs damage sperm membrane integrity, causing a high frequency of head-to-tail or tail-to-tail membrane fusion, whereas this occurrence in the presence of nonpermeating CPA was less than 3%. Finally, the results of experiments 1 and 2 were combined in a mathematical model to predict Gly and EG addition and removal in the presence of SM extender, which would prevent mouse sperm membrane damage. These predictions indicated that, for ICR sperm, both Gly and EG may be added and removed in a single step. However, for B6C3F1 spermatozoa, Gly required a two-step addition while EG only required a single step. For removal from B6C3F1 sperm, Gly required a three-step removal process while EG required a two-step removal.

Mechanism of extracellular ubiquitination in the mammalian epididymis

Posttranslational modification by ubiquitination marks defective or outlived intracellular proteins for proteolytic degradation by the 26S proteasome. The ATP‐dependent, covalent ligation and formation of polyubiquitin chains on substrate proteins requires the presence and activity of a set of ubiquitin activating and conjugating enzymes. While protein ubiquitination typically occurs in the cell cytosol or nucleus, defective mammalian spermatozoa become ubiquitinated on their surface during post‐testicular sperm maturation in the epididymis, suggesting an active molecular mechanism for sperm quality control. Consequently, we hypothesized that the bioactive constituents of ubiquitin‐proteasome pathway were secreted in the mammalian epididymal fluid (EF) and capable of ubiquitinating extrinsic substrates. Western blotting indeed detected the presence of the ubiquitin‐activating enzyme E1 and presumed E1‐ubiquitin thiol–ester intermediates, ubiquitin‐carrier enzyme E2 and presumed E2‐ubiquitin thiol–ester intermediates and the ubiquitin C‐terminal hydrolase PGP 9.5/UCHL1 in the isolated bovine EF. Thiol–ester assays utilizing recombinant ubiquitin‐activating and ubiquitin‐conjugating enzymes, biotinylated substrates, and isolated bovine EF confirmed the activity of the ubiquitin activating and conjugating enzymes within EF. Ubiquitinated proteins were found to be enriched in the defective bull sperm fraction and appropriate proteasomal deubiquitinating and proteolytic activities were measured in the isolated EF by specific fluorescent substrates. The apocrine secretion of cytosolic proteins was visualized in transgenic mice and rats expressing the enhanced green fluorescent protein (eGFP) under the direction of ubiquitin‐C promoter. Accumulation of eGFP, ubiquitin and proteasomes was detected in the apical blebs, the apocrine secretion sites of the caput epididymal epithelia of both the rat and mouse epididymal epithelium, although region‐specific differences exist. Secretion of eGFP and proteasomes continued during the prolonged culture of the isolated rat epididymal epithelial cells in vitro. This study provides evidence that the activity of the ubiquitin system is not limited to the intracellular environment, contributing to a greater understanding of the sperm maturation process during epididymal passage. J. Cell. Physiol. 215: 684–696, 2008.

Development of transgenic rats producing human β-amyloid precursor protein as a model for Alzheimer's disease: Transgene and endogenous APP genes are regulated tissue-specifically

BackgroundAlzheimers disease (AD) is a devastating neurodegenerative disorder that affects a large and growing number of elderly individuals. In addition to idiopathic disease, AD is also associated with autosomal dominant inheritance, which causes a familial form of AD (FAD). Some instances of FAD have been linked to mutations in the β-amyloid protein precursor (APP). Although there are numerous mouse AD models available, few rat AD models, which have several advantages over mice, have been generated.ResultsFischer 344 rats expressing human APP driven by the ubiquitin-C promoter were generated via lentiviral vector infection of Fischer 344 zygotes. We generated two separate APP-transgenic rat lines, APP21 and APP31. Serum levels of human amyloid-beta (Aβ)40 were 298 pg/ml for hemizygous and 486 pg/ml for homozygous APP21 animals. Serum Aβ42 levels in APP21 homozygous rats were 135 pg/ml. Immunohistochemistry in brain showed that the human APP transgene was expressed in neurons, but not in glial cells. These findings were consistent with independent examination of enhanced green fluorescent protein (eGFP) in the brains of eGFP-transgenic rats. APP21 and APP31 rats expressed 7.5- and 3-times more APP mRNA, respectively, than did wild-type rats. Northern blots showed that the human APP transgene, driven by the ubiquitin-C promoter, is expressed significantly more in brain, kidney and lung compared to heart and liver. A similar expression pattern was also seen for the endogenous rat APP. The unexpected similarity in the tissue-specific expression patterns of endogenous rat APP and transgenic human APP mRNAs suggests regulatory elements within the cDNA sequence of APP.ConclusionThis manuscript describes the generation of APP-transgenic inbred Fischer 344 rats. These are the first human AD model rat lines generated by lentiviral infection. The APP21 rat line expresses high levels of human APP and could be a useful model for AD. Tissue-specific expression in the two transgenic rat lines and in wild-type rats contradicts our current understanding of APP gene regulation. Determination of the elements that are responsible for tissue-specific expression of APP may enable new treatment options for AD.

Motility and acrosomal integrity comparisons between electro-ejaculated and epididymal ram sperm after exposure to a range of anisosmotic solutions, cryoprotective agents and low temperatures.

Effective ram sperm cryopreservation protocols, which would yield acceptable lambing rates following artificial insemination (AI), are currently lacking. The objectives of the current studies were to compare the effects of various anisosmotic conditions, cryoprotective agents (CPAs) and chilling on the motility and acrosomal integrity of electro-ejaculated and epididymal ram sperm. Three experiments were conducted. In experiment 1, ejaculated and epididymal ram sperm were exposed to 75, 150, 225, 600, 900 and 1200 milliosmolal (mOsm)/kg sucrose solutions, held for 5 min and then returned to isosmotic condition. Motility characteristics of sperm during exposure to each anisosmotic solutions and after returning to isosmotic conditions were determined. In experiment 2, ejaculated and epididymal ram sperm were exposed to 1M glycerol (Gly), dimethyl sulfoxide (DMSO), ethylene glycol (EG) and propylene glycol (PG) for 5 min and then returned to isosmotic conditions. Motility characteristics of sperm samples during exposure to each CPA solution and after returning to isosmotic conditions were determined. In experiment 3, effects of various temperatures on motility characteristics of ejaculated and epididymal ram sperm were determined after exposing them to three different sub-physiologic temperatures (4, 10 and 22 degrees C) for 30 min and subsequently returning them to 37 degrees C. The motility of ejaculated ram sperm was significantly more affected from anisosmotic stress than was epididymal ram sperm (P<0.05). While anisosmotic stress had no effects on acrosomal integrity of epididymal ram sperm, there was a significant reduction in acrosomal integrity for ejaculated ram sperm after the addition and removal of a 75 mOsm sucrose solution. The abrupt addition and removal of 1M Gly, DMSO, EG or PG had no effect on the motility and acrosomal integrity of epididymal ram sperm (P>0.05). However, there was a slight decrease in acrosomal integrity for ejaculated ram sperm after exposure to 1M Gly, DMSO or EG (P>0.05). Both epididymal and ejaculated ram sperm exhibited temperature-dependent loss of motility and acrosomal integrity (P<0.05). However, ejaculated ram sperm was more sensitive to chilling stress than epididymal sperm (P<0.05). In conclusion, the current data suggest that while epididymal ram sperm is extremely resilient to various cryobiologically relevant stress conditions, ejaculated ram sperm demonstrate greater sensitivity to such stressors. These findings should be taken into account when developing cryopreservation protocols for ejaculated and epididymal ram sperm.

Vitrification of human embryos subjected to blastomere biopsy for pre-implantation genetic screening produces higher survival and pregnancy rates than slow freezing

PurposeCryopreservation of blastocysts, especially those subjected to the trauma due to blastomere biopsy for the purposes of pre-implantation genetic screening (PGS), requires significant optimization. Laboratory and clinical outcomes were compared to determine the effect of two different cryopreservation techniques on the development of human pre-implantation embryos that underwent blastomere biopsy and blastocoel drainage prior to cryopreservation.DesignRetrospective clinical study.Patient(s)Women who requested cryotransfer of supernumerary blastocysts were analyzed by FISH.ResultsThe main outcome measures were post-thaw survival (SR), pregnancy (PR), and implantation (IR). The SR of slowly frozen blastocysts was 83% compared to 97% for vitrified blastocysts. In 160 cases where biopsied embryos were cryotransferred, the results for slowly frozen versus vitrified blastocysts were: SR (71% vs. 95%), PR (23% vs. 37%), and IR (26% vs. 36%, P < 0.05), respectively.ConclusionThe results revealed that vitrified blastocysts provided higher SR, PR and IR as compared to slowly frozen counterparts.

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

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.

Method for detection and identification of multiple chromosomal integration sites in transgenic animals created with lentivirus

Transgene delivery systems, particularly those involving retroviruses, often result in the integration of multiple copies of the transgene throughout the host genome. Since site-specific silencing of trangenes can occur; it becomes important to identify the number and chromosomal location of the multiple copies of the transgenes in order to correlate inheritance of the transgene at a particular chromosomal site with a specific and robust phenotype. Using a technique that combines restriction endonuclease digest and several rounds of PCR amplification followed by nucleotide sequencing, it is possible to identify multiple chromosomal integration sites in transgenic founder animals. By designing genotyping assays to detect each individual integration site in the offspring of these founders, the inheritance of transgenes integrated at specific chromosomal locations can be followed efficiently as the transgenes randomly segregate in subsequent generations. Phenotypic characteristics can then be correlated with inheritance of a transgene integrated at a particular chromosomal location to allow rational selection of breeding animals in order to establish the transgenic line.

Genome resource banking of biomedically important laboratory animals

Genome resource banking is the systematic collection, storage, and redistribution of biomaterials in an organized, logistical, and secure manner. Genome cryobanks usually contain biomaterials and associated genomic information essential for progression of biomedicine, human health, and research. In that regard, appropriate genome cryobanks could provide essential biomaterials for both current and future research projects in the form of various cell types and tissues, including sperm, oocytes, embryos, embryonic or adult stem cells, induced pluripotent stem cells, and gonadal tissues. In addition to cryobanked germplasm, cryobanking of DNA, serum, blood products, and tissues from scientifically, economically, and ecologically important species has become a common practice. For revitalization of the whole organism, cryopreserved germplasm in conjunction with assisted reproductive technologies, offer a powerful approach for research model management, as well as assisting in animal production for agriculture, conservation, and human reproductive medicine. Recently, many developed and developing countries have allocated substantial resources to establish genome resources banks which are responsible for safeguarding scientifically, economically, and ecologically important wild type, mutant, and transgenic plants, fish, and local livestock breeds, as well as wildlife species. This review is dedicated to the memory of Dr. John K. Critser, who has made profound contributions to the science of cryobiology and establishment of genome research and resources centers for mice, rats, and swine. Emphasis will be given to application of genome resource banks to species with substantial contributions to the advancement of biomedicine and human health.

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

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. Mol. Reprod. Dev. 79: 613–625, 2012.

Animal models got you puzzled?: think pig.

Swine are an excellent large animal model for human health and disease because their size and physiology are similar to humans, in particular, with respect to the skin, heart, gastrointestinal tract, and kidneys. In addition, the pig has many emerging technologies that will only enhance the development of the pig as the nonrodent biomedical model of choice.