الكتاب أود أن أشكر الدكتور ليونيد فولكوف واريك بوشار للحصول على المشورة التقنية بشأن المجهر epifluorescence. الدعم المالي بتمويل من قبل المعاهد الكندية لأبحاث الصحة (منحة # MOP-93781) لGB

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R., Zhao, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roles+of+transition+nuclear+proteins+in+spermiogenesis.">Roles of transition nuclear proteins in spermiogenesis.</a> Chromosoma. 111 (8), 483-488 (2003).</li><li>Gr&#233;goire, M. -. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Male-driven+de+novo+mutations+in+haploid+germ+cells.">Male-driven de novo mutations in haploid germ cells.</a> Mol. Hum. Reprod. 19 (8), 495-499 (2013).</li><li>Simard, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Instability+of+trinucleotidic+repeats+during+chromatin+remodeling+in+spermatids.">Instability of trinucleotidic repeats during chromatin remodeling in spermatids.</a> Hum. Mutat. , (2014).</li><li>Wykes, S. M., Krawetz, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+of+spermatogenic+cells+from+adult+transgenic+mouse+testes+using+unit-gravity+sedimentation.">Separation of spermatogenic cells from adult transgenic mouse testes using unit-gravity sedimentation.</a> Mol. Biotechnol. 25 (2), 131-138 (2003).</li><li>Yoshida, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+first+round+of+mouse+spermatogenesis+is+a+distinctive+program+that+lacks+the+self-renewing+spermatogonia+stage.">The first round of mouse spermatogenesis is a distinctive program that lacks the self-renewing spermatogonia stage.</a> Development (Cambridge, England). 133 (8), 1495-1505 (2006).</li><li>Moreno, R. D., Lizama, C., Urz&#250;a, N., Vergara, S. P., Reyes, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caspase+activation+throughout+the+first+wave+of+spermatogenesis+in+the+rat.">Caspase activation throughout the first wave of spermatogenesis in the rat.</a> Cell Tissue Res. 325 (3), 533-540 (2006).</li><li>Meistrich, M. L., Trostle-Weige, P. K., Van Beek, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+of+specific+stages+of+spermatids+from+vitamin+A-synchronized+rat+testes+for+assessment+of+nucleoprotein+changes+during+spermiogenesis.">Separation of specific stages of spermatids from vitamin A-synchronized rat testes for assessment of nucleoprotein changes during spermiogenesis.</a> Biol. Reprod. 51 (2), 334-344 (1994).</li><li>van Pelt, A. M., de Rooij, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synchronization+of+the+seminiferous+epithelium+after+vitamin+A+replacement+in+vitamin+A-deficient+mice.">Synchronization of the seminiferous epithelium after vitamin A replacement in vitamin A-deficient mice.</a> Biol. Reprod. 43, 363-367 (1990).</li><li>Getun, I. V., Torres, B., Bois, P. R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry+purification+of+mouse+meiotic+cells.">Flow cytometry purification of mouse meiotic cells.</a> J. Vis. Exp. , (2011).</li><li>Kovtun, I. V., McMurray, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trinucleotide+expansion+in+haploid+germ+cells+by+gap+repair.">Trinucleotide expansion in haploid germ cells by gap repair.</a> Nature Genet. 27 (4), 407-411 (2001).</li><li>Bastos, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometric+characterization+of+viable+meiotic+and+postmeiotic+cells+by+Hoechst+33342+in+mouse+spermatogenesis.">Flow cytometric characterization of viable meiotic and postmeiotic cells by Hoechst 33342 in mouse spermatogenesis.</a> Cytometry A. 65 (1), 40-49 (2005).</li><li>Lassalle, B., Ziyyat, A., Testart, J., Finaz, C., Lef&#232;vre, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometric+method+to+isolate+round+spermatids+from+mouse+testis.">Flow cytometric method to isolate round spermatids from mouse testis.</a> Hum. Reprod. 14 (2), 388-394 (1999).</li></ol>

The authors declare no competing financial interests.

الخلايا المنوية دائما تحديا للدراسة نظرا لتعقيد ظهارة المنوية، وكذلك النجاح المحدود للثقافة في المختبر. على مر السنين، وقد وضعت الخلايا الجرثومية من الأنواع المختلفة العديد من الطرق لتنقية. تقنيات الترسيب باستخدام تنقية الجاذبية مع Percoll أو الأبقار مصل الزلال ?...

Haploid round spermatids differentiate into spermatozoa by a process called spermiogenesis. This involves many different steps including the acquisition of a flagellum, chromatin and cytoskeleton remodeling, condensation of the nucleus as well as the loss of most of the cytoplasm. These unique cellular events must be finely regulated in order to produce a mature functional gamete with an intact genome suitable for fertilization. Spermiogenesis can hardly be studied in vitro since no reliable cell culture system has so far been able to support progression through the different steps of the process. Moreover, actual in vitro techniques lead to a poor yield1,2. In vivo, proper transitions through the different steps of spermiogenesis are crucial for the natural functional integrity of the male gamete. Successful purification of spermatids according to their differentiation steps has never been accomplished with a level of enrichment sufficient to allow molecular characterization of spermiogenesis. For instance, purification of key steps of the spermatidal differentiation would be especially useful to study the developing acrosome, formation of the midpiece3, cell junction dynamics4, RNA dynamics5, chromatin remodeling process6,7 or genomic stability8. Purification of spermatids has been hampered by their progressive morphological transformation, the lack of known stage-specific external biomarkers, and their peculiar shape and size. 
Although most male germ cells display a direct relationship between DNA staining and ploidy (DNA content), we noticed that such positive correlation is no longer applicable to spermatids. This stems from our early observation that seminiferous tubule sections show variable intensity of DNA staining throughout the different spermiogenesis steps. Although DNA staining is consistent with their haploid set of chromosomes from spermiogenesis steps 1 to 7 (round spermatids), we observed a sharp increase in fluorescence intensity with DAPI or SYTO 16 around the onset of nuclear reorganization and chromatin remodeling (spermiogenesis step 8) reaching a peak at the onset of nuclear condensation (spermiogenesis steps 11-12). Following condensation of the nucleus, DNA staining intensity decreases until spermiation (spermiogenesis step 16). We surmised that this was likely associated with the formation of their peculiar chromatin structure transition where histones are replaced by protamines. We therefore developed a reliable flow cytometry method that allows the separation of spermatids using the variation of DNA intensity of spermatids as a main selection parameter.
A simple flow cytometry approach is described to separate mouse spermatids with high purity (95-100%) based on their apparent DNA content (SYTO16 staining), size and granulosity. Spermatids are separated into four populations; spermiogenesis steps 1-9, 10-12, 13-14 and 15-16. Purified spermatids are suitable for genetic/genomic analysis, as well as proteomic applications as described in a recent publication from our group9.

<table><tbody><tr><td>Isoflurane</td><td>ABBOT</td><td>05260-05</td><td>For mouse anesthesia before euthanasia</td></tr><tr><td>Fetal bovine serum</td><td>Wisent</td><td>90150</td><td>For tube coating</td></tr><tr><td>1x PBS</td><td></td><td></td><td></td></tr><tr><td>EDTA</td><td>BioShop</td><td>EDT</td><td>For sorting buffer preparation</td></tr><tr><td>HEPES</td><td>Sigma</td><td>H</td><td>For sorting buffer preparation</td></tr><tr><td>100% Ethanol</td><td>Les alcools de commerce</td><td>092-09-11N</td><td>For cell fixation</td></tr><tr><td>SYTO 16</td><td>Life Technologies</td><td>S7578</td><td>DNA staining</td></tr><tr><td>5 ml polypropylene round bottom tubes</td><td>BD Falcon</td><td>352063</td><td>Sorted cells collection</td></tr><tr><td>15 ml polypropylene conical bottom tubes</td><td>PROgene</td><td>1500</td><td></td></tr><tr><td>50 ml polypropylene conical bottom tubes</td><td>PROgene</td><td>5000</td><td></td></tr><tr><td>TEC4 anaesthetic vaporizer</td><td>Ohmeda</td><td>1160526</td><td>For mouse euthanasia</td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; gas tank</td><td>Praxair</td><td>C799117902</td><td>For mouse euthanasia</td></tr><tr><td>O&lt;sub&gt;2&lt;/sub&gt; gas tank</td><td>Praxair</td><td>O254130501</td><td>For mouse euthanasia</td></tr><tr><td>Homemade mouse gas chamber</td><td></td><td></td><td>For mouse euthanasia</td></tr><tr><td>40 &amp;micro;m Falcon cell strainer</td><td>Corning Incorporated</td><td>352340</td><td></td></tr><tr><td>50 &amp;mu;m sample line filters</td><td>BD Biosciences</td><td>649049</td><td></td></tr><tr><td>Vortex mixer</td><td>Labnet international, inc.</td><td>S0200</td><td>For cell fixation</td></tr><tr><td>Dynac centrifuge</td><td>Clay Adams</td><td>101</td><td></td></tr><tr><td>Celltrics 50 &amp;micro;m filters</td><td>Partec</td><td>04-004-2327</td><td></td></tr><tr><td>488 nm laser-euipped cell sorter</td><td>BD Biosciences</td><td>FACSAria III</td><td></td></tr><tr><td>Accudop Fluorescent Beads</td><td>BD Biosciences</td><td>345249</td><td></td></tr><tr><td>Sorting Buffer: 1x PBS, 1 mM EDTA pH 8.0, 25 mM HEPES pH 7.0, 1% FBS</td><td></td><td></td><td>FBS is heat-inactivated. Make fresh solution, 0.22 &amp;mu;m filtered and keep at 4&amp;deg;C.</td></tr></tbody></table>

step-specific sorting of mouse spermatids by flow cytometry

تمايز spermatids الفأر هو عملية حاسمة واحدة لإنتاج الأمشاج الذكور وظيفية مع الجينوم سليمة إلى أن تنتقل إلى الجيل التالي. حتى الآن، وقد أعاق الدراسات الجزيئية لهذا التحول الصرفي بسبب عدم وجود طريقة السماح للفصل الكافي لهذه الخطوات الهامة للتمايز النطف للتحليلات لاحقة. المحاولات السابقة في النابضة السليم لهذه الخلايا باستخدام التدفق الخلوي قد يكون صعبا بسبب زيادة غريبة في مضان DNA في spermatids تمر ونين التجديد. وبناء على هذه الملاحظة، ونحن نقدم تفاصيل تدفق بسيط الخلوي المخطط، مما يسمح تنقية استنساخه من أربعة سكان spermatids الماوس ثابتة مع الإيثانول، يمثل كل دولة مختلفة في عملية إعادة عرض النووية. وأكد تخصيب السكان الذين يستخدمون علامات خطوة محددة ومعيارا المورفولوجية. وspermatids النقاء يمكن استخدامها لالجينومية وproteomيحلل جيم.

استراتيجية النابضة استخدامها مع التدفق الخلوي الشكل 1 يمثل استراتيجية النابضة المستخدمة في التدفق الخلوي لفرز أربعة السكان النطف نقية للغاية. لفترة وجيزة، والخلايا مع تلطيخ DNA الإيج?...

Watch this Scientific Journal Video about Step-specific Sorting of Mouse Spermatids by Flow Cytometry at JoVE.com

Step-specific Sorting of Mouse Spermatids by Flow Cytometry

We describe a sorting strategy for mouse spermatids using flow cytometry. Spermatids are sorted into four highly pure populations, including round (spermiogenesis steps 1-9), early elongating (spermiogenesis steps 10-12), late elongating (spermiogenesis steps 13-14) and elongated spermatids (spermiogenesis steps 15-16). DNA staining, size and granulosity are used as selection parameters.

خطوة محددة الفرز ماوس Spermatids بواسطة التدفق الخلوي

The differentiation of mouse spermatids is one critical process for the production of a functional male gamete with an intact genome to be transmitted to ...

step-specific-sorting-of-mouse-spermatids-by-flow-cytometry

Research

JoVE Journal

Developmental Biology

10.5K Views.  Université de Sherbrooke. We describe a sorting strategy for mouse spermatids using flow cytometry. Spermatids are sorted into four highly pure populations, including round (spermiogenesis steps 1-9), early elongating (spermiogenesis steps 10-12), late elongating (spermiogenesis steps 13-14) and elongated spermatids (spermiogenesis steps 15-16). DNA staining, size and granulosity are used as selection parameters.

Video: Step-specific Sorting of Mouse Spermatids by Flow Cytometry

Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by Electroporation

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair cells are aligned in one row of inner hair cells and three rows of outer hair cells that extend along the basal to apical axis of the cochlea. The explant culture technique described here provides an efficient method to isolate and maintain cochlear explants from the embryonic mouse inner ear. Also, the morphology and molecular characteristics of sensory hair cells and nonsensory supporting cells within the cochlear explant cultures resemble those observed in vivo and can be studied within its intrinsic cellular environment. The cochlear explants can serve as important experimental tools for the identification and characterization of molecular and genetic pathways that are involved in cellular specification and patterning. Although transgenic mouse models provide an effective approach for gene expression studies, a considerable number of mouse mutants die during embryonic development thereby hindering the analysis and interpretation of developmental phenotypes. The organ of Corti from mutant mice that die before birth can be cultured so that their in vitro development and responses to different factors can be analyzed. Additionally, we describe a technique for electroporating embryonic cochlear explants ex vivo which can be used to downregulate or overexpress specific gene(s) and analyze their potential endogenous function and test whether specific gene product is necessary or sufficient in a given context to influence mammalian cochlear development1-8.

We present a method that describes isolation and culture of cochlear explants from embryonic mouse inner ear. We also demonstrate a method for gene transfer into cochlear explants via square-wave electroporation. The in vitro explant culture coupled with gene transfer technique enables researchers to study the effects of altering gene expression during development.

ثقافة الجنينية الماوس القوقعة إإكسبلنتس وجين نقل من قبل Electroporation لل

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair ...

A Standardized Approach for Multispecies Purification of Mammalian Male Germ Cells by Mechanical Tissue Dissociation and Flow Cytometry

Fluorescence-activated cell sorting (FACS) has been one of the methods of choice to isolate enriched populations of mammalian testicular germ cells. Currently, it allows the discrimination of up to 9 murine germ cell populations with high yield and purity. This high-resolution in discrimination and purification is possible due to unique changes in chromatin structure and quantity throughout spermatogenesis. These patterns can be captured by flow cytometry of male germ cells stained with fluorescent DNA-binding dyes such as Hoechst-33342 (Hoechst). Herein is a detailed description of a recently developed protocol to isolate mammalian testicular germ cells. Briefly, single cell suspensions are generated from testicular tissue by mechanical dissociation, double stained with Hoechst and propidium iodide (PI) and processed by flow cytometry. A serial gating strategy, including the selection of live cells (PI negative) with different DNA content (Hoechst intensity), is used during FACS sorting to discriminate up to 5 germ cell types. These include, with corresponding average purities (determined by microscopy evaluation): spermatogonia (66%), primary (71%) and secondary (85%) spermatocytes, and spermatids (90%), further separated into round (93%) and elongating (87%) subpopulations. Execution of the entire workflow is straightforward, allows the isolation of 4 cell types simultaneously with the appropriate FACS machine, and can be performed in less than 2 h. As reduced processing time is crucial to preserve the physiology of ex vivo cells, this method is ideal for downstream high-throughput studies of male germ cell biology. Moreover, a standardized protocol for multispecies purification of mammalian germ cells eliminates methodological sources of variables and allows a single set of reagents to be used for different animal models.

This work describes the standardization of a method to obtain purified germ cell populations from testicular tissue of different mammalian species. It is a straightforward protocol that combines mechanical testis dissociation, staining with Hoechst-33342 and propidium iodide, and FACS sorting, with wide applications in comparative studies of male reproductive biology.

نهج موحد لتنقية مولتبيسيسيز من الثدييات خلايا الجرثومية الذكور من الأنسجة الميكانيكية التفكك والتدفق الخلوي

Fluorescence-activated cell sorting (FACS) has been one of the methods of choice to isolate enriched populations of mammalian testicular germ cells. ...

A Seminiferous Tubule Squash Technique for the Cytological Analysis of Spermatogenesis Using the Mouse Model

Meiotic progression in males is a process that requires the concerted action of a number of highly regulated cellular events. Errors occurring during meiosis can lead to infertility, pregnancy loss or genetic defects. Commencing at the onset of puberty and continuing throughout adulthood, continuous semi-synchronous waves of spermatocytes undergo spermatogenesis and ultimately form haploid sperm. The first wave of mouse spermatocytes undergoing meiotic initiation appear at day 10 post-partum (10 dpp) and are released into the lumen of seminiferous tubules as mature sperm at 35 dpp. Therefore, it is advantageous to utilize mice within this developmental time-window in order to obtain highly enriched populations of interest. Analysis of rare cell stages is more difficult in older mice due to the contribution of successive spermatogenic waves, which increase the diversity of the cellular populations within the tubules. The method described here is an easily implemented technique for the cytological evaluation of the cells found within the seminiferous tubules of mice, including spermatogonia, spermatocytes, and spermatids. The tubule squash technique maintains the integrity of isolated male germ cells and allows examination of cellular structures that are not easily visualized with other techniques. To demonstrate the possible applications of this tubule squash technique, spindle assembly was monitored in spermatocytes progressing through the prophase to metaphase I transition (G2/MI transition). In addition, centrosome duplication, meiotic sex chromosome inactivation (MSCI), and chromosome bouquet formation were assessed as examples of the cytological structures that can be observed using this tubule squash method. This technique can be used to pinpoint specific defects during spermatogenesis that are caused by mutation or exogenous perturbation, and thus, contributes to our molecular understanding of spermatogenesis.

The goal of this tubule squash technique is to rapidly assess cytological features of developing mouse spermatocytes while preserving cellular integrity. This method allows for the study of all stages of spermatogenesis, and can be easily implemented alongside other biochemical and molecular biological approaches for the study of mouse meiosis.

تقنية إسكواش أنبوب سيمينيفيروس لتحليل الحيوانات المنوية باستخدام نموذج الماوس الخلوية

Meiotic progression in males is a process that requires the concerted action of a number of highly regulated cellular events. Errors occurring during ...

Enrichment of Pachytene Spermatocytes and Spermatids from Mouse Testes Using Standard Laboratory Equipment

To characterize each step of spermatogenesis, researchers must separate different subpopulations of germ cells from testes. However, isolating discrete populations is challenging, because the adult testis contains a complex mix of germ cells from all steps of spermatogenesis along with certain populations of somatic cells. Over the past few decades, different techniques such as centrifugal elutriation, fluorescence-activated cell sorting (FACS), and STA-PUT have been successfully applied to the isolation of germ cells. A drawback is that they all require dedicated devices and specialized training. Following principles underlying the STA-PUT method, a simple protocol has been developed for the isolation of pachytene spermatocytes, round spermatids, and elongating spermatids from mouse testes. After preparing a single cell suspension of testicular cells, specific cell populations are enriched by gravity sedimentation through a discontinuous bovine serum albumin (BSA) density gradient. The cell fractions are then manually collected and microscopically analysed. This modified density gradient for round spermatids (MDR) sedimentation protocol can be widely applied, because it requires only standard laboratory equipment. Furthermore, the protocol requires minimal starting materials, reducing its cost and use of laboratory animals.

Presented here is a protocol for enriching pachytene spermatocytes, round spermatids, and elongating spermatids from adult mouse testes using a discontinuous bovine serum albumin density gradient with standard laboratory equipment.

إثراء الخلايا المنوية والبطفومنات من اختبار الماوس باستخدام معدات المختبرات القياسية

To characterize each step of spermatogenesis, researchers must separate different subpopulations of germ cells from testes. However, isolating discrete ...

Isolation of Murine Spermatogenic Cells using a Violet-Excited Cell-Permeable DNA Binding Dye

Isolation of meiotic spermatocytes is essential to investigate molecular mechanisms underlying meiosis and spermatogenesis. Although there are established cell isolation protocols using Hoechst 33342 staining in combination with fluorescence-activated cell sorting, it requires cell sorters equipped with an ultraviolet laser. Here we describe a cell isolation protocol using the DyeCycle Violet (DCV) stain, a low cytotoxicity DNA binding dye structurally similar to Hoechst 33342. DCV can be excited by both ultraviolet and violet lasers, which improves the flexibility of equipment choice, including a cell sorter not equipped with an ultraviolet laser. Using this protocol, one can isolate three live-cell subpopulations in meiotic prophase I, including leptotene/zygotene, pachytene, and diplotene spermatocytes, as well as post-meiotic round spermatids. We also describe a protocol to prepare single-cell suspension from mouse testes. Overall, the procedure requires a short time to complete (4-5 hours depending on the number of needed cells), which facilitates many downstream applications.

Here we present a simple and efficient method to isolate live meiotic and post-meiotic germ cells from adult mouse testes. Using a low-cytotoxicity, violet-excited DNA binding dye and fluorescence-activated cell sorting, one can isolate highly enriched spermatogenic cell populations for many downstream applications.

عزل الخلايا المنوية مورين باستخدام البنفسجي-متحمس خلية نفاذة صبغة ملزمة

Isolation of meiotic spermatocytes is essential to investigate molecular mechanisms underlying meiosis and spermatogenesis. Although there are established ...

Flow Cytometry Purification of Mouse Meiotic Cells

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the unique differentiation programs that are manifest during meiosis. Two principal methods have been developed to purify, to varying degrees, various meiotic fractions from both adult and immature animals: elutriation or Staput (sedimentation) using BSA and/or percoll gradients. Both of these methods rely on cell size and density to separate meiotic cells1-5. Overall, except for few cell populations6, these protocols fail to yield sufficient purity of the numerous meiotic cell populations that are necessary for detailed molecular analyses. Moreover, with such methods usually one type of meiotic cells can be purified at a given time, which adds an extra level of complexity regarding the reproducibility and homogeneity when comparing meiotic cell samples.
Here, we describe a refined method that allows one to easily visualize, identify, and purify meiotic cells, from germ cells to round spermatids, using FACS combined with Hoechst 33342 staining7,8. This method provides an overall snapshot of the entire meiotic process and allows one to highly purify viable cells from most stage of meiosis. These purified cells can then be analyzed in detail for molecular changes that accompany progression through meiosis, for example changes in gene expression9,10and the dynamics of nucleosome occupancy at hotspots of meiotic recombination11.

An efficient method to obtain highly purified viable meiotic fractions from mouse testis is described, which combines a refined cell dissociation protocol with fluorescent activated cell sorting (FACS). This method takes advantage of differences in the DNA content and nuclear density of discrete meiotic fractions.

تدفق تنقية الخلوي للخلايا الفأر الانتصافي

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the ...

Biology

<meta charset="utf8"></head><body>إجراء حقن المنوية المستديرة الفئران لدراسة التطور الجنيني

Mouse Round Spermatid Injection

Round spermatids, characterized by their haploid genetic content, represent the precursor cells to mature spermatozoa. Through the innovative technique of round spermatid injection (ROSI), oocytes can be successfully fertilized and developed into viable fetuses. In a groundbreaking milestone achieved in 1995, the first mouse fetus was born through ROSI technology. ROSI has since emerged as a pivotal tool for unraveling the intricate mechanisms governing embryonic development and holds significant potential in various applications, including the acceleration of mouse generation and the production of genetically modified mice. In 1996, a milestone was reached when the first human fetus was born through ROSI technology. However, the clinical applications of this method have shown a fluctuating pattern of success and failure. To date, ROSI technology has not found widespread application in clinical practice, primarily due to its low birth efficiency and insufficient validation of fetal safety. This article provides a comprehensive account of the precise methods of performing ROSI in mice, aiming to shed new light on basic research and its potential clinical applications.

<meta charset="utf8"></head><body>حقن الفأر المستدير Spermatid

Here, we introduce a method for performing round spermatid injection (ROSI) in mice, a technique with promising clinical applications and utility for investigating the mechanisms underlying embryonic development.

حقن الفأر المستدير Spermatid

Round spermatids, characterized by their haploid genetic content, represent the precursor cells to mature spermatozoa. Through the innovative technique of ...

Simple and Efficient Technique for the Preparation of Testicular Cell Suspensions

Mammalian testes are very complex organs that contain over 30 different cell types, including somatic testicular cells and different stages of germline cells. This heterogeneity is an important drawback concerning the study of the bases of mammalian spermatogenesis, as pure or enriched cell populations in certain stages of sperm development are needed for most molecular analyses1.
 Various strategies such as Staput2,3, centrifugal elutriation1, and flow cytometry (FC)4,5 have been employed to obtain enriched or purified testicular cell populations in order to enable differential gene expression studies. 
It is required that cells are in suspension for most enrichment/ purification approaches. Ideally, the cell suspension will be representative of the original tissue, have a high proportion of viable cells and few multinucleates - which tend to form because of the syncytial nature of the seminiferous epithelium6,7 - and lack cell clumps1 . Previous reports had evidenced that testicular cell suspensions prepared by an exclusively mechanical method clumped more easily than trypsinized ones1 . On the other hand, enzymatic treatments with RNAses and/or disaggregating enzymes like trypsin and collagenase lead to specific macromolecules degradation, which is undesirable for certain downstream applications. The ideal process should be as short as possible and involve minimal manipulation, so as to achieve a good preservation of macromolecules of interest such as mRNAs. Current protocols for the preparation of cell suspensions from solid tissues are usually time-consuming, highly operator-dependent, and may selectively damage certain cell types1,8 . 
	The protocol presented here combines the advantages of a highly reproducible and extremely brief mechanical disaggregation with the absence of enzymatic treatment, leading to good quality cell suspensions that can be used for flow cytometric analysis and sorting4, and ulterior gene expression studies9 .

A novel protocol for the mechanical preparation of testicular cell suspensions from rodent material, avoiding enzymes and detergents, is described. The method is very simple, fast, reproducible, and renders good quality cell suspensions, which are suitable for flow sorting and RNA extraction.

تقنيات بسيطة وفعالة لإعداد تعليق خلية الخصية

Mammalian testes are very complex organs that contain over 30 different cell types, including somatic testicular cells and different stages of germline ...

خطوة محددة الفرز ماوس Spermatids بواسطة التدفق الخلوي

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ثقافة الجنينية الماوس القوقعة إإكسبلنتس وجين نقل من قبل Electroporation لل

نهج موحد لتنقية مولتبيسيسيز من الثدييات خلايا الجرثومية الذكور من الأنسجة الميكانيكية التفكك والتدفق الخلوي

تقنية إسكواش أنبوب سيمينيفيروس لتحليل الحيوانات المنوية باستخدام نموذج الماوس الخلوية

إثراء الخلايا المنوية والبطفومنات من اختبار الماوس باستخدام معدات المختبرات القياسية

عزل الخلايا المنوية مورين باستخدام البنفسجي-متحمس خلية نفاذة صبغة ملزمة

تدفق تنقية الخلوي للخلايا الفأر الانتصافي

حقن الفأر المستدير Spermatid

حقن الفأر المستدير Spermatid

حقن الفأر المستدير Spermatid

تقنيات بسيطة وفعالة لإعداد تعليق خلية الخصية