Cansu Agca

Transcriptional Profiling of Pig Embryogenesis by Using a 15-K Member Unigene Set Specific for Pig Reproductive Tissues and Embryos

Abstract Differential mRNA expression patterns were evaluated between germinal vesicle oocytes (pgvo), four-cell (p4civv), blastocyst (pblivv), and in vitro-produced four-cell (p4civp) and in vitro-produced blastocyst (pblivp) stage embryos to determine key transcripts responsible for early embryonic development in the pig. Five comparisons were made: pgvo to p4civv, p4civv to pblivv, pgvo to pblivv, p4civv to p4civp, and pblivv to pblivp. ANOVA (P < 0.05) was performed with the Benjamini and Hochberg false-discovery-rate multiple correction test on each comparison. A comparison of pgvo to p4civv, p4civv to pblivv, and pgvo to pblivv resulted in 3214, 1989, and 4528 differentially detected cDNAs, respectively. Real-time PCR analysis on seven transcripts showed an identical pattern of changes in expression as observed on the microarrays, while one transcript deviated at a single cell stage. There were 1409 and 1696 differentially detected cDNAs between the in vitro- and in vivo-produced embryos at the four-cell and blastocyst stages, respectively, without the Benjamini and Hochberg false-discovery-rate multiple correction test. Real-time polymerase chain reaction (PCR) analysis on four genes at the four-cell stage showed an identical pattern of gene expression as found on the microarrays. Real-time PCR analysis on four of five genes at the blastocyst stage showed an identical pattern of gene expression as found on the microarrays. Thus, only 1 of the 39 comparisons of the pattern of gene expression exhibited a major deviation between the microarray and the real-time PCR. These results illustrate the complex mechanisms involved in pig early embryonic development.

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.

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.

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.

Various Physical Stress Factors on Rat Sperm Motility, Integrity of Acrosome, and Plasma Membrane

The objective of this study was to determine the effects of various physical interventions such as centrifugation regimes, Percoll gradient separation, and repeated pipetting on various viability parameters of epididymal sperm of Fischer 344 (F-344) and Sprague-Dawley (SD) rat strains. Three experiments were conducted. In experiment 1, sperm motility and acrosomal and membrane integrity were compared after exposing sperm samples to 200, 400, 600, and 800 x g centrifugal forces for 5, 10, or 15 minutes. In experiment 2, sperm motility and acrosomal and membrane integrity were compared after passing them through a Percoll separation using centrifugal forces of 600, 800, 1000, and 1200 x g for either 15 or 30 minutes. In experiment 3, the effect of repeated pipetting (2, 4, 6, 8, and 10 times) on motility and membrane integrity of rat sperm was compared with that on mouse, ram, bull, and boar sperm. The results revealed that both F-344 and SD rat sperm motility and membrane integrity were significantly affected by centrifugation (P < .05). The acrosomal integrity of SD rat sperm was affected after using 800 x g centrifugation force for 10 or 15 minutes (P < .05), whereas F-344 rat sperm acrosomal integrity was not affected by any centrifugation regimes (P > .05). Sperm from SD rats also had higher motility and membrane integrity loss than did sperm from F-344 rats after centrifugation and pipetting (P < .05). Percoll gradient separation did not cause significant motility loss or acrosomal damage to either F-344 or SD sperm (P > .05). Repeated pipetting had a dramatic adverse effect on both rat and mouse sperm motility (P < .05) as compared with sperm from bull, boar, and ram, which were not affected at all (P > .05). These data suggest that rat sperm have unique properties that need to be considered during centrifugation, Percoll gradient separation, and pipetting procedures.

Expression of mitochondrial transcription factor A (TFAM) during porcine gametogenesis and preimplantation embryo development.

Mitochondrial transcription factor A (TFAM) is responsible for stability, maintenance, and transcriptional control of mitochondrial DNA (mtDNA). We have studied the expression and distribution of TFAM in the gametes and preimplantation embryos of the domestic pig (Sus scrofa). We hypothesized that TFAM is not present in the boar sperm mitochondria to reduce the possibility of paternal mtDNA propagation in the progeny. In contrast, we anticipated that Tfam gene is expressed in a developmental stage‐dependent manner in porcine oocytes and embryos. The appropriate TFAM band of 25 kDa was detected by Western blotting in ejaculated boar spermatozoa, as well as in porcine oocytes and zygotes. Boar sperm extracts also displayed several bands >25 kDa suggestive of post‐translational modification by ubiquitination, confirmed by affinity purification of ubiquitinated proteins. TFAM immunoreactivity was relegated to the sperm tail principal piece and sperm head in fully differentiated spermatozoa. The content of Tfam mRNA increased considerably from the germinal vesicle to blastocyst stage and also between in vitro fertilized and cultured blastocysts compared to in vivo‐derived blastocysts. TFAM protein accumulated in the oocytes during maturation and was reduced by proteolysis after fertilization. This pattern was not mirrored in parthenogenetically activated oocytes and zygotes reconstructed by SCNT, suggesting deviant processing of TFAM protein and transcript after oocyte/embryo manipulation. Thus, TFAM may exert a critical role in porcine gametogenesis and preimplantation embryo development. Altogether, our data on the role of TFAM in mitochondrial function and inheritance have broad implications for cell physiology and evolutionary biology. J. Cell. Physiol. 217: 529–543, 2008.

Large-scale generation and analysis of expressed sequence tags from porcine ovary.

Abstract One method to identify the factors that control ovarian function is to characterize the genes that are expressed in ovary. In the present study, cDNA libraries from fetal, neonatal, and prepubertal porcine ovaries, pubertal ovaries on different days of the estrous cycle (Days 0 [follicle], 5, and 12 [follicle and corpus luteum]), and follicles isolated from weaned sows (diameter, 2, 4, 6, and 8 mm) were constructed and sequenced. A total of 22 176 cDNAs were sequenced, of which 15 613 were of sufficient quality for clustering. Clustering of cDNAs resulted in 8507 contigs, 6294 (74%) of which were comprised of a single sequence. Sixty-eight percent of the contigs had consensus sequences that were homologous to existing Tentative Consensus (TC) sequences or mature transcripts (ET) in The Institute for Genomic Research Porcine Gene Index. The consensus sequences were classified according to the Gene Ontology Index. Most cDNA-encoded proteins were components of the nucleus, ribosome, or mitochondrion. The proteins primarily functioned in binding, catalysis, and transport. Nearly 75% of the proteins were involved in metabolism and cell growth and/or maintenance. Analysis of the cDNA frequency across different libraries demonstrated differential gene expression within different-size follicles, between follicles and corpora lutea, and across developmental time-points. The expression of selected genes (analyzed by ribonuclease protection assay and Northern blotting) was consistent with the frequency of their respective cDNA in the individual libraries. This porcine ovary unigene set will be useful for identifying factors and mechanisms controlling ovarian follicular development in a variety of species.

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

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.

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

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 (Gtrt), GnRH + eCG (G + Etrt), and GnRH + eCG + hCG (G + E + Htrt) 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, Gtrt, and G + Etrt; however, over 2,600 genes were significantly different between NC and G + E + Htrt (P < 0.05). Genes upregulated in G + E + Htrt 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 + Htrt 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 (Gtrt) and ovarian hyper stimulation (G + Etrt) had minimal effects on gene expression, whereas induction of ovulation (G + E + Htrt) caused major changes in gene expression of rat COCs. This study provides comprehensive information about regulated genes during late follicle development and ovulation induction. Mol. Reprod. Dev.