Sperm‐Binding Assay Using an In Vitro 3D Model of the Mammalian Cumulus‐Oocyte Complex

2020 ◽  
Vol 86 (1) ◽  
Author(s):  
Julieta Gabriela Hamze ◽  
María Jiménez‐Movilla ◽  
Raquel Romar
Keyword(s):  
3D Model ◽  
Binding Assay ◽  
Sperm Binding ◽  
Cumulus Oocyte Complex

149 DEVELOPMENT OF AN IN VITRO BIOLUMINESCENT SPERM BINDING ASSAY: PRELIMINARY DATA

2014 ◽  
Vol 26 (1) ◽  
pp. 188
Author(s):  
R. C. Youngblood ◽  
S. T. Willard ◽  
P. L. Ryan ◽  
J. M. Feugang
Keyword(s):  
Epithelial Cells ◽  
Quantum Dot ◽  
Reproductive Tract ◽  
Binding Assay ◽  
Agricultural Research ◽  
Detection System ◽  
Female Reproductive Tract ◽  
Uterine Epithelial Cells ◽  
Sperm Binding

Quantum dot technology has enabled researchers to incorporate the intrinsic properties of such nanoparticles into physiological exploration. Previous work from our laboratory has demonstrated that quantum dots can be incorporated into spermatozoa without deleterious effects to physiological parameters such as motility, viability, and fertilizing potential (Feugang et al. 2012). However, the journey of spermatozoa within the female reproductive tract is met with many physicochemical obstacles and checkpoints that include the binding of spermatozoa to utero-oviducal epithelial cells. Moreover, the binding ability/affinity of quantum dot-labelled spermatozoa has not been tested and therefore, the objective of this study is to test the binding semblance of quantum dot-labelled spermatozoa to uterine epithelial cells compared to normal sperm, and the subsequent use of the technology to develop a bioluminescent sperm binding assay. Porcine uterine epithelial (PUE) cells were seeded into 96-well clear-bottomed plates (20 000 cells/well) and allowed to grow to 95% confluency. Motile spermatozoa were selected from fresh pooled semen of fertile boars and labelled with quantum dot nanoparticles to form quantum sperm, as previously described (Feugang et al. 2012). Final concentrations of 107 labelled (QD+) and non-labelled (QD–) spermatozoa were added to monolayers of PUE cells and co-incubated in PBS/polyvinylpyrrolidone (PVP) at 37°C, 5% CO2. The control consisted of PUE cells alone in the PBS/PVP medium. Each treatment was performed in triplicate and experiments were repeated 3 times. After 1 h of co-incubation, the supernatant from each well was transferred to the adjacent three wells. The co-incubated wells containing expected PUE-sperm binding were then washed 3 times with PBS/PVP to eliminate any unbound sperm. PUE-quantum sperm (QD+) and PUE-non-labelled sperm (QD–) complexes were verified using bright field microscopy, followed by measurement of photonic emission from each well (GloMax Multi Detection System, Promega, Madison, WI, USA). Data was analysed by ANOVA with the threshold of significance fixed at P < 0.05. There were no visual differences in binding patterns between QD+ and QD–, which appeared similar under the microscope. However, the photonic signals (relative luminescent units; RLU) from QD+ wells were significantly higher than both the control and QD– wells (2534.84 ± 639.91 v. 542.46 ± 639.91 and 806.48 ± 639.91 RLU; P < 0.05). Supernatants collected from the QD+ wells, representing unbound quantum sperm, had the highest photonic emissions when compared to all other wells, with or without spermatozoa (19 948.23 ± 639.91 RLU; P < 0.05). Results demonstrate that quantum dot nanoparticles can be incorporated into boar spermatozoa without affecting their binding affinity to uterine epithelial cells, and their subsequent use in a biophotonic sperm binding assay. Further optimization and experimentations are ongoing to establish whether bioluminescent quantum sperm could serve to develop sensitive in vitro binding assays to better characterise sperm viability. Support was provided by U.S. Department of Agriculture Agricultural Research Service (USDA-ARS) grant number 58-6402-3-0120

P–567 Re-establishment of fertilization competency of the oocyte via CRISPR/dCas9 epigenome edition technology

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
G N Sahin ◽  
G Soyler ◽  
A Kayabolen ◽  
A Kocabay ◽  
A C Taskın ◽  
...  
Keyword(s):  
Western Blotting ◽  
Binding Assay ◽  
Gene Activation ◽  
Surface Protein ◽  
Fold Increase ◽  
High Dose ◽  
Sperm Binding ◽  
Fertilization Capacity

Abstract Study question Is it possible to increase the decreased levels of the sperm-oocyte binding protein, Juno, to restore the fertilization capacity of the oocyte, via the use of the CRISPR/dCas9 activation system? Summary answer JUNO domain (in the oocyte) suppressed with melamine was opened using the CRISPR/dCas9 system and sperm-oocyte binding and fertilization capability have been restored. What is known already Melamine is a chemical that is widely used in the manufacture of laminates, plastics, etc. Evidence reveals that long-term exposure to melamine could damage reproductive systems in mammals leading to infertility. Izumo1 is the only cell surface protein expressed on sperm that is known to be essential for sperm-egg interaction in vivo. It was in vitro shown that high-dose feeding of melamine to female mice led to a significant decrease of JUNO on the plasma membrane of eggs. CRISPR/dCas9 system can provide the gene activation or repression to activate the target gene via Sam and Tet1 based systems. Study design, size, duration Six different gRNAs were designed for the transfection of oocytes. Six-week-old mice were fed with melamine (50mg/kg/day) for 2 weeks via gavage. Melamine gavage, water gavage, and control groups (n = 15 /each group) were created. CRISPR activation plasmids (SAM) were given by piezo microinjection into the GV oocytes (n = 100 oocytes/each group). Fertilization capacity was evaluated by sperm binding assay, qPCR, Western blotting, and IF staining. Two technical replicates were used in molecular studies. Participants/materials, setting, methods 293T cells were transfected (dCas9 SAM plasmids+gRNA) with Fugene. Mice randomly were assigned to 3 groups (n = 15), as each was given orally a dose of 50mg/kg/d of melamine, only water or no water via gavage. Microinjection of plasmids was performed. Post-injection, oocytes were incubated until MII stage. For binding and fertilization evaluation, motile sperms were incubated with oocytes, and pronuclei were checked. Juno and IZUMO1 levels were evaluated by qPCR, Western blotting, and IF staining. Main results and the role of chance As the SAM system is more efficient compared to the Tet1 system when tested in 293t cells, the SAM system was used for mouse experiments. As a result of qPCR performed in oocytes collected at the end of gavage, it was observed that the JUNO expression levels were decreased by 40 folds in melamine fed mice (p &lt; 0.05). The decrease in the level of JUNO protein was demonstrated by IF stainings, and a decrease in the oocyte count along with abnormal uterine shapes was also observed in this group. IZUMO1 expression in motile sperms was demonstrated by qPCR before sperm binding assay and the position of the IZUMO 1 domain before the acrosome reaction was demonstrated by IF stainings. Sperm binding assay has demonstrated a 70% reduction in fertilization competency of melamine-treated oocytes (p &lt; 0.05). SAM plasmids and JUNO gRNA were given to oocytes by piezo injection. By sperm binding experiments conducted to evaluate fertilization capacities after microinjection, it was shown that the fertilization capacity was restored by 75% (p &lt; 0.05). Re-gaining of Juno expression in the oocytes was supported by a 60 fold increase in qPCR results. Recovery of JUNO protein expression in the oocytes was also demonstrated by IF stainings. Limitations, reasons for caution There is no known promoter region for the JUNO gene in the mouse. Therefore, we designed gRNAs targeting possible promoter regions. However, we have used two activation systems(SAM and Tet1) that are widely used to open a closed gene, but other activation systems (acetylation, etc.) can also be tried. Wider implications of the findings: This study is valuable since: -it presents a possible cure for unsuccessful fertilization in related cases. -it possibly reveals melamine’s unknown way of action. - it presents a new approach as a sperm-binding assay to be used in IVF clinics since Juno can also be expressed in somatic cell lines. Trial registration number non-clinical trials

Evaluation of the function of fresh and frozen - thawed sex-sorted and non-sorted stallion spermatozoa using a heterologous oocyte binding assay

2010 ◽  
Vol 22 (4) ◽  
pp. 710 ◽  
Author(s):  
J. R. Clulow ◽  
G. Evans ◽  
W. M. C. Maxwell ◽  
L. H. A. Morris
Keyword(s):  
Zona Pellucida ◽  
Binding Assay ◽  
Binding Ability ◽  
Treatment Groups ◽  
Functional Integrity ◽  
Sperm Binding ◽  
Heterologous Sperm ◽  
Bovine Oocytes ◽  
Better Than

The aim of the present study was to evaluate the potential oocyte binding ability and functional integrity of fresh or frozen–thawed, sex-sorted or non-sorted stallion spermatozoa. In the absence of effective IVF procedures in the horse, a heterologous sperm-binding assay was used as an indicator of fertilising capacity to assess differences in the ability of stallion spermatozoa to bind to bovine oocytes. The functional integrity of four treatment groups was assessed: (1) fresh non-sorted spermatozoa; (2) fresh sex-sorted spermatozoa; (3) frozen–thawed non-sorted spermatozoa; and (4) frozen–thawed sex-sorted spermatozoa. Spermatozoa found in association with the zona pellucida of the bovine oocytes were deemed ‘attached’ or ‘bound’ depending on their characterisation as either acrosome intact or acrosome reacted, respectively. Significantly less frozen–thawed spermatozoa were found attached to the oocytes compared with fresh spermatozoa. No significant differences were identified between the number of attached sex-sorted and non-sorted frozen–thawed spermatozoa. However, significantly more sex-sorted than non-sorted fresh spermatozoa were found attached to the oocytes after 1 h coincubation, although after 3 h coincubation this difference was no longer apparent. In conclusion, sex-sorted fresh and frozen–thawed stallion spermatozoa are functionally capable of attaching and binding to bovine oocytes in vitro. Furthermore, fresh sex-sorted spermatozoa attach better than non-sorted spermatozoa, suggesting that they have a more advanced capacitation-like status.

Generation of a 3D model to better mimic NAFLD in vitro

2016 ◽  
Vol 54 (12) ◽  
pp. 1343-1404
Author(s):  
LS Spitzhorn ◽  
MA Kawala ◽  
J Adjaye
Keyword(s):  
3D Model

Differences between antigenic determinants of pig and cat zona pellucida proteins

Reproduction ◽  
2000 ◽  
pp. 15-23 ◽  
Author(s):  
K Jewgenow ◽  
M Rohleder ◽  
I Wegner
Keyword(s):  
In Vitro Fertilization ◽  
Zona Pellucida ◽  
Antigenic Determinants ◽  
Vitro Fertilization ◽  
Contraceptive Vaccine ◽  
Sperm Binding ◽  
Dependent Inhibition ◽  
Dose Dependent Inhibition ◽  
Dose Dependent

Despite many efforts, the control of reproduction in feral cat populations is still a problem in urban regions around the world. Immunocontraception is a promising approach; thus the present study examined the suitability of the widely used pig zona pellucida proteins (pZP) for contraception in feral domestic cats. Purified zona pellucida proteins obtained from pig and cat ovaries were used to produce highly specific antisera in rabbits. Antibodies against pZP raised in rabbits or lions were not effective inhibitors of either in vitro sperm binding (cat spermatozoa to cat oocytes) or in vitro fertilization in cats, whereas antibodies against feline zona pellucida proteins (fZP) raised in rabbits showed a dose-dependent inhibition of in vitro fertilization. Immunoelectrophoresis, ELISA and immunohistology of ovaries confirmed these results, showing crossreactivity of anti-fZP sera to fZP and to a lesser extent to pZP, but no interaction of anti-pZP sera with fZP. It is concluded that cat and pig zonae pellucidae express a very small number of shared antigenic determinants, making the use of pZP vaccine in cats questionable. A contraceptive vaccine based on feline zona pellucida determinants will be a better choice for the control of reproduction in feral cats if immunogenity can be achieved.

Glucocorticoid metabolism in bovine cumulus-oocyte complex during in vitro maturation

2014 ◽  
Author(s):  
Masafumi Tetsuka ◽  
Ryo Takagi ◽  
Nobuhiro Ambo ◽  
Yuta Zempo ◽  
Asuka Onuma
Keyword(s):  
In Vitro Maturation ◽  
Cumulus Oocyte Complex ◽  
Glucocorticoid Metabolism

In vitro validation of a 3-dimensional transrectal ultrasound system for prostate biopsiess

2007 ◽  
Vol 30 (4) ◽  
pp. 77
Author(s):  
Derek Cool ◽  
Shi Sherebrin ◽  
Jonathan Izawa ◽  
Joseph Chin ◽  
Aaron Fenster
Keyword(s):  
Prostate Biopsy ◽  
3D Model ◽  
Transrectal Ultrasound ◽  
Surface Topology ◽  
Sufficient Information ◽  
Needle Guidance ◽  
3 Dimensional ◽  
High Incidence ◽  
3D Information

Introduction: Transrectal ultrasound (TRUS) prostate biopsy (Bx) is currently confined to 2D information to both target and record 3D Bx locations. Accurate placement of Bx needles cannot be verified without 3D information, and recording Bx sites in 2D does not provide sufficient information to accurately guide the high incidence of repeat Bx. We have designed a 3D TRUS prostate Bx system that augments the current 2D TRUS system and provides tools for biopsy-planning, needle guidance, and recording of the biopsy core locations entirely in 3D. Methods: Our Bx system displays a 3D model of the patient’s prostate, which is generated intra-procedure from a collection of 2D TRUS images, representative of the particular prostate shape. Bx targets are selected, needle guidance is facilitated, and 3D Bx sites are recorded within the 3D context of the prostate model. The complete 3D Bx system was validated, in vitro, by performing standard ten-core Bx on anatomical phantoms of two patient’s prostates. The accuracy of the needle-guidance, Bx location recording, and 3D model volume and surface topology were validated against a CT gold standard. Results: The Bx system successfully reconstructed the 3D patient prostate models with a mean volume error of 3.2 ± 7.6%. Using the 3D system, needles were accurately guided to the pre-determined targets with a mean error of 2.26 ± 1.03 mm and the 3D locations of the Bx cores were accurately recorded with a mean distance error of 1.47 ± 0.79 mm. Conclusion: We have successfully developed a 3D TRUS prostate biopsy system and validated the system in vitro. A pilot study has been initiated to apply the system clinically.

Coumarin-Fatty Acid Conjugates as Potential ERα/AKT-1 Antagonists for ER Positive Breast Cancer

2020 ◽  
Vol 20 (4) ◽  
pp. 437-449
Author(s):  
Jubie Selvaraj ◽  
Jameera B.A. John ◽  
Nanjan M. Joghee ◽  
Justin Antony ◽  
Ashish Wadhwani ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Gene Expression ◽  
Fatty Acid ◽  
Binding Assay ◽  
Uterine Cancer ◽  
Design Development ◽  
Structure Based Drug Design ◽  
Ic50 Values ◽  
Cancer Activity

Background: : Current drugs used for the treatment of hormone-dependent breast cancer function as anti-estrogens in the breast, in addition to Estrogen Receptor (ER) agonists in the uterus, thus elevate a woman’s risk of developing uterine cancer. This is due to the lack of selective binding and partial agonistic effect of these drugs towards estrogen receptors. In recent years, therefore, researchers have turned their attention towards antiestrogens devoid of these agonist properties and thus have a mechanism of action different from the existing drugs. Objective:: In this context, we report here the design, development and in vitro evaluation of some novel pharmacophores containing coumarin and fatty acid scaffolds for their anti-breast cancer activity. Methods: : A library of coumarin-fatty acid conjugates was designed using structure-based drug design approach. The conjugates which have shown good in silico results were then synthesized, characterized and evaluated for their anti-breast cancer activity by MTT assay, Apoptotic assay, Cell proliferation assay, Estrogen binding assay and Gene expression study. Results: Out of the fifteen compounds screened, two compounds, SAC-2 and LNAC-2, showed good activity with IC50 values 22µg/ml, 25μg/ml, respectively. These compounds suppressed the proliferation of ER overexpressed MCF-7 cells, increased ERα degradation and hence inactivate the ERα pathway. ER binding assay and gene expression RT-PCR study reveal that SAC-2 downregulated the expression of ERα receptor and AKT-1 gene. Conclusion:: Compound SAC-2 is a good antagonist to ER and hence has a potential for treating breast cancer and other cancers where AKT plays an important role.

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