Fluorescence- and magnetic-activated cell sorting strategies to separate spermatozoa involving plural contributors from biological mixtures for human identification

Xu, Yan; Xie, Jianhui; Chen, Ronghua; Cao, Yu; Ping, Yuan; Xu, Qiantong; Hu, Wei; Wu, Dan; Gu, Li-Hua; Zhou, Heng; Chen, Xin; Zhao, Ziqin; Zhong, Jun; Li, Rui

doi:10.1038/srep36515

Cited by 25 publications

(23 citation statements)

References 29 publications

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“…Smaller numbers of sperm cells can also be isolated accurately using this method. Less than 60 sperm cells are needed to obtain a complete STR profile using optical tweezers whereas methods requiring antibodies have been shown to require approximately 10 5 cells [10]. Optical tweezers also do not require the use of antibodies to successfully separate sperm and epithelial cells so complications regarding antibody binding specificity or efficiency do not exist.…”

Section: Discussionmentioning

confidence: 99%

“…However, the optical tweezer equipment can be modified to view fluorescence (i.e. with an appropriate dichroic mirror, see for example [28–32]; consequently, sperm cell separation using antibody-based methods (such as that recently reported by Xu et al [10]) could also be modified for use in optical trapping if needed, delivering cell separation not only by morphology but also antibody differentiation.…”

Section: Discussionmentioning

confidence: 99%

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Optical tweezers as an effective tool for spermatozoa isolation from mixed forensic samples

et al. 2019

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A single focus optical tweezer is formed when a laser beam is launched through a high numerical aperture immersion objective. This objective focuses the beam down to a diffraction-limited spot, which creates an optical trap where cells suspended in aqueous solutions can be held fixed. Spermatozoa, an often probative cell type in forensic investigations, can be captured inside this optical trap and dragged one by one across millimeter-length distances in order to create a cluster of cells which can be subsequently drawn up into a capillary for collection. Sperm cells are then ejected onto a sterile cover slip, counted, and transferred to a tube for DNA analysis workflow. The objective of this research was to optimize sperm cell collection for maximum DNA yield, and to determine the number of trapped sperm cells necessary to produce a full STR profile. A varying number of sperm cells from both a single-source semen sample and a mock sexual assault sample were isolated utilizing optical tweezers and processed using conventional STR analysis methods. Results demonstrated that approximately 50 trapped spermatozoa were required to obtain a consistently full DNA profile. A complete, single-source DNA profile was also achieved by isolating sperm cells via optical trapping from a mixture of sperm and vaginal epithelial cells. Based on these results, optical tweezers are a viable option for forensic applications such as separation of mixed populations of cells in forensic evidence.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Optical tweezers as an effective tool for spermatozoa isolation from mixed forensic samples

et al. 2019

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“…Additionally, fluorescent dyes tagged for FACS and particular antibodies for MACS may influence normal cellular physiology and functions 18 , 19 . For these reasons, label-free single-cell analysis techniques have attracted considerable attention because the complexity of sample preparation and analysis procedures is relatively low and intrinsic physical cell properties such as cell size, shape, compressibility, and polarizability can be measured while minimizing the effect on cell physiology and function 19 – 21 .…”

Section: Introductionmentioning

confidence: 99%

Label-free analysis of the characteristics of a single cell trapped by acoustic tweezers

Kim

Park

Lim

et al. 2017

Sci Rep

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Single-cell analysis is essential to understand the physical and functional characteristics of cells. The basic knowledge of these characteristics is important to elucidate the unique features of various cells and causative factors of diseases and determine the most effective treatments for diseases. Recently, acoustic tweezers based on tightly focused ultrasound microbeam have attracted considerable attention owing to their capability to grab and separate a single cell from a heterogeneous cell sample and to measure its physical cell properties. However, the measurement cannot be performed while trapping the target cell, because the current method uses long ultrasound pulses for grabbing one cell and short pulses for interrogating the target cell. In this paper, we demonstrate that short ultrasound pulses can be used for generating acoustic trapping force comparable to that with long pulses by adjusting the pulse repetition frequency (PRF). This enables us to capture a single cell and measure its physical properties simultaneously. Furthermore, it is shown that short ultrasound pulses at a PRF of 167 kHz can trap and separate either one red blood cell or one prostate cancer cell and facilitate the simultaneous measurement of its integrated backscattering coefficient related to the cell size and mechanical properties.

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“…Functionally, FS provides a scaffold for glycolytic and signaling enzymes during capacitation that is important for hyperactivated motility [62–64]. AKAP3 has been reported to localize only to the principal piece region of mouse sperm [65, 66]. In human sperm, antibodies against variants of AKAP3 have detected disparate localization patterns in head and tail structures [45, 67].…”

Section: Resultsmentioning

confidence: 99%

Human OVGP1 enhances tyrosine phosphorylation of proteins in the fibrous sheath involving AKAP3 and increases sperm-zona binding

Zhao

Kan

2019

J Assist Reprod Genet

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Purpose To investigate if the recombinant human oviduct-specific glycoprotein (rHuOVGP1)–enhanced tyrosine-phosphorylated (pY) proteins are components of specific structure(s) of the sperm tail and if rHuOVGP1 binds to the oocyte and enhances sperm-egg binding. Methods Immunofluorescent staining and confocal microscopy were performed to examine the localization of pY proteins, outer dense fiber (ODF), and A-Kinase Associated Protein 3 (AKAP3) in human sperm during capacitation. Western blot and immunoprecipitation were employed to analyze protein levels of pY proteins and AKAP3. Immunofluorescent staining was performed to examine the binding of rHuOVGP1 to human oocytes. The effect of rHuOVGP1 on enhancing sperm-zona binding was examined using hemizona assay. Results pY proteins were detected mainly in the fibrous sheath (FS) surrounding the ODF with a relatively weak immunoreaction in the neck and mid-piece. Western blot analysis revealed co-migration of the pY 105 kDa protein with AKAP3, which was further confirmed by immunoprecipitation correlating immunofluorescent results of co-localization of pY proteins with AKAP3 in the sperm tail. rHuOVGP1 binds specifically to the zona pellucida (ZP) of human oocytes. Prior incubation of sperm and/or ZP with rHuOVGP1 increased sperm-egg binding. Conclusions The present study revealed that one of the major rHuOVGP1-enhanced pY proteins could be AKAP3 of the FS and that rHuOVGP1 is capable of binding to human ZP and its presence in the medium results in an increase in sperm-zona binding. Supplement of rHuOVGP1 in in vitro fertilization media could be beneficial for enhancement of the fertilizing ability of human sperm.

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Fluorescence- and magnetic-activated cell sorting strategies to separate spermatozoa involving plural contributors from biological mixtures for human identification

Cited by 25 publications

References 29 publications

Optical tweezers as an effective tool for spermatozoa isolation from mixed forensic samples

Optical tweezers as an effective tool for spermatozoa isolation from mixed forensic samples

Label-free analysis of the characteristics of a single cell trapped by acoustic tweezers

Human OVGP1 enhances tyrosine phosphorylation of proteins in the fibrous sheath involving AKAP3 and increases sperm-zona binding

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