227. Eggs with a surprise: the 'sperm-specific' protein SPRASA is also expressed in the oocyte

2008 ◽  
Vol 20 (9) ◽  
pp. 27
Author(s):  
L. W. Chamley ◽  
A. Wagner ◽  
D. Prendergast ◽  
K. J. Woad ◽  
A. N. Shelling
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
In Silico Analysis ◽  
Human Sperm ◽  
Cumulus Cells ◽  
Reproductive Organs ◽  
Pcr Analysis ◽  
Quantitative Real Time Pcr ◽  
Bovine Oocytes

SPRASA is a newly identified protein which in silico analysis suggests is not expressed in other tissues. Antibodies reactive with SPRASA have been identified in some infertile men and an antiserum reactive with recombinant SPRASA prevented human sperm binding to hamster oocytes in vitro, indicating an important role in sperm/oocyte recognition. The aim of this study was to investigate the spatial and temporal expression of SPRASA in reproductive and other tissues. Brain, thymus, heart, spleen, kidney, liver and the reproductive organs from duplicate female and male Balb/C mice were collected at several postnatal timepoints. RNA was extracted, reversed transcribed and analysed by quantitative real time PCR for SPRASA expression. Abattoir-derived, in vitro matured, bovine oocytes were examined for SPRASA expression by fluorescent immunochemistry. To examine SPRASA binding sites on oocytes, matured bovine oocytes were exposed to biotinylated recombinant human SPRASA or biotinylated α-lactalbumin (control), then visualised by confocal microscopy using DTAF-conjugated streptavidin. We found SPRASA mRNA was expressed in the reproductive organs of both females and male mice from postnatal day 10. Fluorescent immunochemistry indicated SPRASA was expressed on the oolemmal membrane and in the few cumulus cells remaining attached to zona-intact oocytes. Control preimmune serum did not stain the oocytes or cumulus cells. Recombinant human SPRASA bound to the oolemmal membrane of both zona intact and zona free bovine oocytes. To date the expression of SPRASA has only been reported in the testes/sperm with an additional single EST identified in brain. Our quantitative real-time PCR analysis demonstrated SPRASA is also expressed in the female reproductive organs. This was confirmed by our immunoassays which show oocytes and possibly cumulus cells express SRPASA while the oolemmal membrane has the ability to bind (sperm-derived) SPRASA. That SPRASA expression is restricted sperm and oocytes confirms the likely function of this protein in reproduction.

scholarly journals The circRAB3IP Mediated by eIF4A3 and LEF1 Contributes to Enzalutamide Resistance in Prostate Cancer by Targeting miR-133a-3p/miR-133b/SGK1 Pathway

2021 ◽  
Vol 11 ◽  
Author(s):  
Dong Chen ◽  
Yaqin Wang ◽  
Feiya Yang ◽  
Adili Keranmu ◽  
Qingxin Zhao ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Real Time ◽  
Expression Pattern ◽  
Real Time Pcr ◽  
Bioinformatic Analysis ◽  
Biological Functions ◽  
Quantitative Real Time Pcr ◽  
Regulatory Effects

An increasing number of studies have shown that circRNAs are closely related to the carcinogenesis and development of prostate cancer (PCa). However, little is known about the effect of the biological functions of circRNAs on the enzalutamide resistance of PCa. Through bioinformatic analysis and experiments, we investigated the expression pattern of circRNAs in enzalutamide-resistant PCa cells. Quantitative real-time PCR was used to detect the expression of circRAB3IP, and plasmids that knock down or overexpress circRAB3IP were used to evaluate its effect on the enzalutamide sensitivity of PCa cells. Mechanistically, we explored the potential regulatory effects of eIF4A3 and LEF1 on the biogenesis of circRAB3IP. Our in vivo and in vitro data indicated that increased expression of circRAB3IP was found in enzalutamide-resistant PCa, and knockdown of circRAB3IP significantly enhanced enzalutamide sensitivity in PCa cells. However, upregulation of circRAB3IP resulted in the opposite effects. Further mechanistic research demonstrated that circRAB3IP could regulate the expression of serum and glucocorticoid-regulated kinase 1 (SGK1) by serving as a sponge that directly targets miR-133a-3p/miR-133b. Then, we showed that circRAB3IP partially exerted its biological functions via SGK1 signaling. Furthermore, we discovered that eIF4A3 and LEF1 might increase circRAB3IP expression in PCa.

scholarly journals Use of quantitative real-time PCR for studying the dissemination of Leptospira interrogans in the guinea pig infection model of leptospirosis

2009 ◽  
Vol 58 (5) ◽  
pp. 648-655 ◽  
Author(s):  
Kristel Lourdault ◽  
Florence Aviat ◽  
Mathieu Picardeau
Keyword(s):  
Guinea Pig ◽  
Real Time ◽  
Real Time Pcr ◽  
Guinea Pigs ◽  
Infection Model ◽  
Avirulent Strain ◽  
Leptospira Interrogans ◽  
Quantitative Real Time Pcr ◽  
Guinea Pig Model

The dynamics of leptospirosis infection have been poorly studied. The purpose of this study was to determine the LD50, rate of bacterial dissemination, histopathology and antibody responses against leptospira following inoculation with the highly virulent Leptospira interrogans Fiocruz L1-130 strain in a guinea pig model of leptospirosis. Three routes of infection (intraperitoneal, conjunctival and subcutaneous inoculation) were used to establish disease in guinea pigs. The size and kinetics of leptospiral burdens in the blood and tissues of infected animals were determined over a 1 week course of infection using quantitative real-time PCR (qPCR). Bacteraemia peaked at day 5 post-infection reaching more than 5×104 leptospires ml−1. The highest spirochaetal load was found in the liver and kidneys, and was associated with alterations in organ tissues and a decline in liver and kidney functions. In contrast, lesions and bacteria were not detected in guinea pigs infected with an avirulent strain derived from a high-passage-number in vitro-passaged variant of the Fiocruz L1-130 strain. The use of qPCR supports the findings of earlier studies and provides an easy and reliable method for the quantification of L. interrogans in the tissues of infected animals. qPCR will be used in future studies to evaluate the efficacy of vaccine candidates against leptospirosis and the virulence of selected L. interrogans mutants relative to the parental strain.

scholarly journals Quantitative real-time PCR analysis and microarray-based RNA expression of HER2 in relation to outcome

2007 ◽  
Vol 18 (5) ◽  
pp. 845-850 ◽  
Author(s):  
J. Bergqvist ◽  
J.F. Ohd ◽  
J. Smeds ◽  
S. Klaar ◽  
J. Isola ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Pcr Analysis ◽  
Rna Expression ◽  
Quantitative Real Time Pcr

195 ALTERED mRNA TRANSCRIPT EXPRESSION OF IN VITRO- VERSUS IN VIVO-MATURED PORCINE OOCYTES

2012 ◽  
Vol 24 (1) ◽  
pp. 210
Author(s):  
L. D. Spate ◽  
B. K. Redel ◽  
K. M. Whitworth ◽  
W. G. Spollen ◽  
S. M. Blake ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Real Time ◽  
Real Time Pcr ◽  
Genetic Background ◽  
Cumulus Cells ◽  
Developmental Competence ◽  
Illumina Genome Analyzer ◽  
Similar Genetic Background

In contrast to oocytes matured in vitro, porcine embryos that result from in vivo maturation and fertilization have a high developmental competence and readily make the transition from oocyte to blastocyst. This observation led us to investigate the transcript profile differences between in vivo- and in vitro-matured porcine oocytes. For the in vivo-matured group, oviducts of 3 gilts of similar genetic background were flushed 2 days after detection of standing oestrus. MII oocytes were collected in pools of 10 and snap frozen in liquid nitrogen for RNA isolation. The in vitro-matured oocytes were obtained by euthanizing 3 gilts, again with a similar genetic background and recovering the ovaries. Follicles (2 to 8 mm in size) were aspirated and oocytes with multiple layers of cumulus cells and uniform cytoplasm were placed in M-199 supplemented with LH, FSH and epidermal growth factor for 42 h. Upon maturation, cumulus cells were stripped and the healthy MII oocytes were collected in pools of 10 and snap frozen. Total RNA was extracted from 3 pools of 10 oocytes for both treatments using an All prep DNA/RNA micro isolation kit (Qiagen, Valencia, CA, USA). Complementary DNA was synthesized using oligo (dT′) primed reverse transcriptase with superscript III (Invitrogen, Carlsbad, CA, USA). Second-strand cDNA was synthesized using DNA polymerase I and sequenced using Illumina Genome Analyzer II. All reads were aligned to a custom-built porcine transcriptome. There were over 18 million reads in the 2 maturation groups that tiled to the 34 433-member transcriptome: 1317 transcripts were detected with a P ≤ 0.1 (Students t-test), a minimum of 7 reads in at least 1 of the treatments and ≥2-fold difference. Real-time PCR was used on selected transcripts. Comparative CT Method was used on an IQ real-time PCR system with the Bio–Rad SYBR green mix. Statistical differences were determined using the Proc general linear model procedure of SAS (SAS Institute Inc., Cary, NC) and means separated with a l.s.d. (P ≤ 0.05). The misrepresented transcripts from the sequencing data were also characterized using the functional annotation tool DAVID. Twelve pathways were overrepresented in the in vitro-matured oocytes (the top 4 are pathways to cancer, spliceosome, cell cycle and ubiquitin-mediated proteolysis). Eight pathways were underrepresented in the in vitro-matured oocytes (the top 4 are cytoskeleton regulation, T-cell receptor signaling pathway, ubiquitin-mediated proteolysis and cell cycle). Eight transcripts were selected for real-time PCR. ZP2 was higher in the in vitro-matured oocytes as determined by both sequencing and real time. ATG4, HSP90, UBAP2 and SOX4 were not different, regardless of assay. SLC7A3, MRPS36 and PDHX2 were not different based on sequencing, but based on real-time MRPS36 and PDHX2, were higher in the in vivo group and SLC7A3 was higher in the in vitro group. In conclusion, there is an abundance of misregulated transcripts and altered pathways in in vitro-matured oocytes. This dataset is a tool that may provide clues to improve the in vitro maturation process so that in vitro-matured oocytes will be more like their in vivo-matured counterparts, thus improving developmental competence. Funded by Food for the 21st Century.

342 EFFECTS OF MELATONIN ON PREIMPLANTATION DEVELOPMENT OF PORCINE PARTHENOGENETIC EMBRYOS

2010 ◽  
Vol 22 (1) ◽  
pp. 327 ◽  
Author(s):  
J.-T. Kang ◽  
O.-J. Koo ◽  
D.-K. Kwon ◽  
S.-J. Park ◽  
M. Atikuzzaman ◽  
...  
Keyword(s):  
Treatment Group ◽  
Beneficial Effect ◽  
Real Time ◽  
Cell Expansion ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Pcr Analysis ◽  
Melatonin Treatment ◽  
Parthenogenetic Embryos

In mammalian species, melatonin is a multi-functional pineal gland hormone that regulates several circadian and seasonal rhythms including reproduction. However, the melatonin study was not common as to the oocytes in the pig. Recently, we reported that exogenous melatonin has beneficial effects on nuclear and cytoplasmic maturation during porcine oocyte IVM and we also reported an existence of melatonin receptor on the cumulus cells and granulose cells (Kang JT et al. 2009 J. Pineal Res. 46(1), 22-28). In this study, as adding further experiments rather than our previous study, we investigated effect of exogeneous melatonin (10 ng mL-1) on the porcine oocytes and analyzed possible factors which can be responsible for that results. Oocytes were recovered by aspiration of slaughterhouse ovaries, and then matured in TCM-199 supplemented with EGF, insulin, pyruvate, cystine, and gonadotropin. Expression of apoptosis-related genes mRNA in oocytes cultured with melatonin were evaluated by real-time PCR (Exp 1), cumulus cell expansion on COC was assessed on the microscopes during in vitro maturation (Exp 2), and developmental effects between melatonin treatement group and non-treatment group on the in vitro culture of parthenogenetically activated oocytes was investigated (Exp 3). In results, oocytes matured with melatonin were assessed for the expression of apoptosis-related genes Bcl-xl (anti-apoptotic gene) and Bax (proapoptotic gene) by real-time quantitative PCR. Analysis of data showed that the expression of Bcl-xl was higher compared to the control while the expression of Bax was decreased relative to the control (P < 0.05). Cumulus cell expansion was evaluated under a stereomicroscope at 22 h, 44 h during IVM. Representative photomicrographs of porcine COC at the start of the IVM, after 22 h and 44 h treatment with melatonin, are shown in Figure. After 22 h of melatonin treatment, cumulus cells were visually expanded compared with non-treatment group. We analyzed significantly greater proportion of parthenogenetically activated oocytes developed to blastocyst when the IVM medium was supplemented with melatonin. Melatonin treatment in the IVM has consequently beneficial effect on the blastocyst formation rates on the development of porcine parthenogenetic embryos (15.4%) compared to non-treatment group (10.7%, P < 0.05). However, cleavage frequency was not affectedby the treatment. In conclusion, the present study demonstrated that melatonin had a beneficial effect on the development of parthenogenetically activated porcine embryos, probably through decreased apoptosis rate and increased cumulus cell expansion. This study was supported by Korean MKE, MEST (BK21 program), and Hanhwa L&C

Evaluation of reference genes for quantitative real-time PCR analysis of gene expression in Hainan medaka (Oryzias curvinotus)

Gene Reports ◽  
2019 ◽  
Vol 14 ◽  
pp. 94-99 ◽  
Author(s):  
Zhongdian Dong ◽  
Pushun Chen ◽  
Ning Zhang ◽  
Shunkai Huang ◽  
Hairui Zhang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Real Time Pcr ◽  
Reference Genes ◽  
Pcr Analysis ◽  
Quantitative Real Time Pcr ◽  
Oryzias Curvinotus

scholarly journals Investigating reference genes for quantitative real-time PCR analysis across four chicken tissues

Gene ◽  
2015 ◽  
Vol 561 (1) ◽  
pp. 82-87 ◽  
Author(s):  
S. Bagés ◽  
J. Estany ◽  
M. Tor ◽  
R.N. Pena
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Reference Genes ◽  
Pcr Analysis ◽  
Quantitative Real Time Pcr ◽  
Chicken Tissues
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