עבודה זו נתמכה על ידי NIGMS (R01GM11755) כדי P.W.J על ידי מלגת גרנט אימונים מ הלאומי סרטן המכון (NIH) (CA009110) ג. ה, ג'יי-אייץ

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H., Ishiguro, K., Kudo, N., Watanabe, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+meiosis-specific+cohesins+in+mouse+embryonic+oocytes.">Studying meiosis-specific cohesins in mouse embryonic oocytes.</a> Methods Mol Biol. 957 (1), 47-57 (2013).</li><li>Susiarjo, M., Rubio, C., Hunt, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analyzing+mammalian+female+meiosis.">Analyzing mammalian female meiosis.</a> Methods Mol Biol. 558, 339-354 (2009).</li><li>Hassold, T., Hunt, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=To+err+(meiotically)+is+human:+the+genesis+of+human+aneuploidy.">To err (meiotically) is human: the genesis of human aneuploidy.</a> Nat Rev Genet. 2 (4), 280-291 (2001).</li><li>Hassold, T., Hall, H., Hunt, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+origin+of+human+aneuploidy:+where+we+have+been,+where+we+are+going.">The origin of human aneuploidy: where we have been, where we are going.</a> Hum Mol Genet. 16, R203-R208 (2007).</li><li>Pincus, G., Enzmann, E. 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המחברים אין לחשוף.

ההכנות התפשטות כרומטין לאפשר לחוקרים ללמוד באופן כרונולוגי הנשי מיוזה יונקים ו לוקליזציה דינמי של חלבונים המעורבים. בכפולות כרומטין עובריים ו neonatal מאפשרים ניתוח קרוב של אירועים ברחבי meiotic prophase. מפה של מפה של השני ואני כרומטין כפולות יכול לשמש כדי להבחין בין האחות יחיד chromatids מ זיווג כרומו?...

במהלך יצירת זרע, סינכרונית למחצה גלי העלייה הגדולים של meiotic בתאי הנבט במהירות ובאופן רצוף מתחדשות ב בזרמה בתחילת גיל ההתבגרות וברחבי לבגרות1. בניגוד הזכרים, מיוזה אצל נקבות מאותחלת אך ורק במהלך התפתחות העובר. בעקבות הלידה, oocytes נשארים נעצר בשלב dictyate ממושך של prophase אני עם שלפוחית-germinal שלם (GV; מעטפת הגרעין) עד גיל ההתבגרות. מיד עם תחילת גיל ההתבגרות, תת-קבוצה של oocytes המזמורים נבחרים לעבור גדילה, התבגרות, סימון אתחול של חידוש meiotic. חידוש meiotic ב oocytes וחוה מתבטאת על ידי היעלמותו של GV בתהליך המכונה התמוטטות germinal שלפוחית (GVBD). Oocyte לציפוי כרומוזום עיבוי, סגרגציה, ואחריו הגוף קוטב ההבלטה. Oocytes הופכים נעצרה על סדר MII, מומרץ כדי להשלים את החטיבה השנייה והאחרונה meiotic רק לאחר ההפריה.
פוריות נשית תלויה מאוד להצלחת meiotic prophase אני התקדמות. המפתח לכל זה היא היווצרות-חיבור פיזי בין כרומוזומים הומולוגיים הידועה בשם chiasmata, אשר מתווך על ידי תיקון המושרה מעברי חוט כפול דנ א (DSBs) באמצעות כבל מוצלב רקומבינציה2. תהליך זה מתרחש בתוך ההקשר של לפיגום עשירים בחלבון דינאמית הידועה בשם המתחם synaptonemal (SC) המהווה בין כרומוזום הומולוגי כדי להקל על שלהם synapsis3. SC הוא דמוי רוכסן tripartite מבנה המורכב שני אלמנטים לרוחב מקבילים המחוברים באמצעות חלבונים באזור המרכז שמחזיק homologs יחד במהלך מתמשך DNA לתקן. לפני synapsis, מבשרי מרכיבי לרוחב, שנקרא אלמנטים צירית, יוצרים בין האחות chromatids. Synaptonemal קומפלקס חלבונים כגון SYCP2 ו- SYCP3 טופס צירית הגורמים colocalize בצירים לכידות כרומטידה אחות במהלך prophase המוקדמות. אלה מאוחר יותר לשמש מחייב אתרים עבור החלבון נימה רוחבי, SYCP1, אשר מאפשר הרכבה יסוד מרכזי, synapsis בין מיושר homologs4. ב oocytes העכבר, synapsis מלאה מסומן על ידי הנוכחות של 20 לגמרי חופפים מתיחות SYCP3 ו SYCP1, אשר ניתן לאבחן באמצעות כרומטין להפיץ את ההכנות. Synapsis הושלמה בעת כניסתו substage pachytene, לפיה ג'יי ג'יי אברהמס בוגרת זה נועדו טופס chiasmata בין homologs מעוטרים mutL הדימרים homolog (MLH1/3) כדי לקדם את עיבוד מדויק שלהם,5,6 , 7. קיום מתחמי כרומוזומים (SMC), כולל cohesin, condensin ו- SMC5/6 מתחם מבניים חשובים עבור הרגולציה של כרומוזום dynamics ומבנה במהלך המיוזה8,9, 10,11,12. באופן קולקטיבי, אירועים אלה להבטיח bi-כיוון נכון של כרומוזומים הומולוגיים מנוגדות הפולנים פלך בעקבות פירוק של SC.
מחזור התא meiotic הוא מודל חזק כדי לבחון את התפקידים של חלבונים שונים בתחזוקת הגנום עקב אינדוקציה מתוכנת תיקון עוקבות של דנ א DSBs. יתר על כן, בתרבית של המיוזה היא גם דוגמנית הרלוונטיים לצורך המחקר של שינויים epigenetic, החתמה13. עם זאת, מבחינה טכנית קשה להעריך אירועים אלה במהלך המיוזה הנשי, אשר מתקיים בעובר ו neonatal השחלות אצל יונקים (איור 1). Prophase אני יכול להיות מחולק 5 substages: leptonema, zygonema, pachynema, diplonema ו- dictyate. במסמך זה, אנו מתארים כיצד לבודד, להבחין בין עוברי ו neonatal השחלות, האשכים (איור 2). הותאם מפעולות שתואר קודם לכן, כתב יד זה גם מתאר פרוטוקול (שלב 1) עם הפגנה וידאו להכנת prophase meiotic הנשי אני כרומטין מתפשט14,15,16,17 . בשילוב עם immunolabelling, כפי שמתואר שלבים 6-7, פרוטוקול זה מאפשר ניתוח מיקרוסקופי מפורט של prophase אני אירועים oocytes.
Oogenesis מועדת לשגיאות, כרומוזום missegregation אירועים במהלך החטיבה meiotic הראשון מייצג את המקור הנפוץ ביותר של מחלה גנטית רומא18,19. כתב יד זה (פרוטוקול שלב 2), נתאר פרוטוקול שבו oocytes ביים-GV בוגרת המחולצים להתקפת השחלות של עכברים הנשי למבוגרים. בתנאים תומכת, oocytes וחוה עוברים הורמון מחלמן תלויית חידוש מיוזה בעקבות בידוד ותרבות20. בעקבות חידוש meiotic, oocytes להתקדמות מיוזה, ואז לעצור-מפה של השני. Oocytes נשארים נעצר ב מפה של השני, אלא אם כן מופרית. שלבים 2-5, אנחנו להסתגל פרוטוקולים שדווחה בעבר עם הפגנה וידאו כדי לתאר כיצד לאסוף, תרבות ולהכין oocytes MI, MII כרומטין להפיץ את ההכנות21. זה כרומטין התפשטות הטכניקה מאפשר immunolabelling ברורה של חלבונים הקשורים כרומוזומים. יתר על כן, פרוטוקול זה יכול לשמש גם כדי להבדיל בין כרומוזום טרינארית ו univalent, ולא ניתן לפתור עוד אחות אחת chromatids כדי להקל על הערכה של oocyte פלואידיות. לכן, בנוסף חשיפת דפוסי לוקליזציה של חלבונים meiotic, פרוטוקול זה יכול לשמש גם כלי רב ערך עבור שחקרתי סיבות פוטנציאליות של כרומוזום missegregation במהלך MI MII.

<table><tbody><tr><td>10X Phosphate buffered saline (PBS) pH 7.4</td><td>Quality Biological</td><td>119-069-161</td><td></td></tr><tr><td>60 mm x 15 mm petri dishes</td><td>Denville</td><td>T1106</td><td></td></tr><tr><td>35mm x 12mm petri dishes</td><td>Denville</td><td>T1103</td><td></td></tr><tr><td>Fine forceps</td><td>VWR</td><td>300-050</td><td></td></tr><tr><td>Micro dissection scissors</td><td>Ted Pella</td><td>1340</td><td></td></tr><tr><td>Dissecting&amp;nbsp;scissors, sharp tip, 41/2&quot;</td><td>VWR</td><td>82027-578</td><td></td></tr><tr><td>Watch glass square 1 5/8</td><td>Carolina Biological Supply Company</td><td>742300</td><td>Autoclave before each use</td></tr><tr><td>Sucrose</td><td>Sigma</td><td>S8501</td><td></td></tr><tr><td>Trisodium Citrate Dihydrate</td><td>Sigma</td><td>S1804</td><td></td></tr><tr><td>EDTA</td><td>Sigma</td><td>E6758</td><td></td></tr><tr><td>Trizma Base</td><td>Sigma</td><td>T1503</td><td></td></tr><tr><td>1,4-Dithiothreitol (DTT)</td><td>Sigma</td><td>646563</td><td>Add to hypotonic buffer immediately before use</td></tr><tr><td>50x Protease Inhibitor</td><td>Roche</td><td>11873580001</td><td>Add to hypotonic buffer immediately before use</td></tr><tr><td>Turberculin syringe with needle (1cc, 27 G x 1/2 in)</td><td>Becton, Dickinson (BD)</td><td>309623</td><td></td></tr><tr><td>Gold seal ultra frost glass slides (25X75mm, 1mm thick)</td><td>Thermo</td><td>3063-002</td><td></td></tr><tr><td>Super PAP pen, large (liquid blocker)</td><td>Electron Microscopy Sciences (EMS)</td><td>71310</td><td></td></tr><tr><td>16% Paraformaldehyde aqueous</td><td>Electron Microscopy Sciences (EMS)</td><td>15710</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>T8787</td><td>Detergent in manuscript</td></tr><tr><td>Immuno stain moisture chamber</td><td>Evergreen</td><td>240-9020-Z10</td><td></td></tr><tr><td>Coplin jars</td><td>Fisher</td><td>08-815</td><td></td></tr><tr><td>Photo-flo 200</td><td>Electron Microscopy Sciences (EMS)</td><td>74257</td><td>Wetting agent in manuscript</td></tr><tr><td>Pregnant mare serum gonadotropin (PMSG)</td><td>Sigma</td><td>G4877</td><td>Store aliquots at -20C</td></tr><tr><td>Human chorionic gonadotropin (HCG)</td><td>Sigma</td><td>C0434</td><td>Store aliquots at -20C</td></tr><tr><td>PYREX Spot Plates with nine concave cavities</td><td>Fisher</td><td>13-748B</td><td>Autoclave before each use</td></tr><tr><td>Glass capillary</td><td>Fisher</td><td>22260943</td><td>For making oocyte collection needles</td></tr><tr><td>Brand Parafilm M</td><td>Sigma</td><td>BR701501</td><td>For making oocyte collection needles</td></tr><tr><td>Latex rubber tubing (3.2 mm inner diameter x 6.4 mm outer diameter)</td><td>Fisher</td><td>14-178-5B</td><td>For making oocyte collection needles</td></tr><tr><td>Latex rubber tubing (6.4 mm inner diameter x 11.1 mm outer diameter)</td><td>Fisher</td><td>14-178-5D</td><td>For making oocyte collection needles</td></tr><tr><td>Flexible silicone rubber nosepiece, hard plastic mouthpiece and 15 inches of latex tubing</td><td>Sigma</td><td>A5177-5EA</td><td>For making oocyte collection needles</td></tr><tr><td>Mitutoyo micrometer head</td><td>Mituoyo</td><td>150-208</td><td>For making oocyte collection needles</td></tr><tr><td>Syringe 5 mL</td><td>BD Biosciences</td><td>309646</td><td>For making oocyte collection needles</td></tr><tr><td>Syringe filter 0.45 &amp;mu;m filter</td><td>Corning</td><td>431220</td><td>For making oocyte collection needles</td></tr><tr><td>Glass Pasteur Pipette 5 3/4&quot;</td><td>Fisher</td><td>13-678-6A</td><td>For making oocyte collection needles</td></tr><tr><td>83 x 0.5 mm pipette tip</td><td>Denville</td><td>P3080</td><td>For making oocyte collection needles</td></tr><tr><td>&amp;alpha;-MEM</td><td>Invitrogen</td><td>11415049</td><td></td></tr><tr><td>Waymouth&#039;s media</td><td>Life Technologies</td><td>11220035</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Fisher</td><td>A3160602</td><td></td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Sigma</td><td>A3311</td><td>For oocyte culture media</td></tr><tr><td>500ml filter units 0.22um</td><td>Denville</td><td>F5227</td><td></td></tr><tr><td>Hyaluronidase</td><td>Sigma</td><td>H3506</td><td></td></tr><tr><td>Tyrode&amp;rsquo;s solution</td><td>Sigma</td><td>T1788</td><td>Store aliquots at -20C</td></tr><tr><td>Horse serum</td><td>Sigma</td><td>H-1270</td><td>For ADB/wash buffer</td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Sigma</td><td>A1470</td><td>For ADB/wash buffer</td></tr><tr><td>VECTASHIELD antifade mounting medium with DAPI</td><td>Vector Labs</td><td>H-1200</td><td></td></tr><tr><td>Microscope cover slides (22mmx60mm)</td><td>Fisher</td><td>12-544-G</td><td></td></tr><tr><td>Clear nail polish</td><td>Amazon</td><td>N/A</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Microscopes&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>SteREO Discovery.V8</td><td>Zeiss</td><td>495015-0001-000</td><td></td></tr><tr><td>Observer Z1</td><td>Zeiss</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Primary Antibodies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Mouse anti-MLH1</td><td>Life Technologies</td><td>MA5-15431</td><td>Dilution factor- 1:100</td></tr><tr><td>Rabbit anti-SYCP1</td><td>Life Technologies</td><td>PA1-16763</td><td>Dilution factor- 1:1000</td></tr><tr><td>Goat anti-SCP3</td><td>Santa Cruz</td><td>sc-20845</td><td>Dilution factor- 1:50</td></tr><tr><td>Human anti-centromere protein</td><td>Antibodies Incorporated</td><td>15-235</td><td>Dilution factor- 1:100</td></tr><tr><td>Rabbit anti- TOPOII</td><td>Abcam</td><td>ab109524</td><td>Dilution factor- 1:100</td></tr><tr><td>Mouse anti-Histone H4 (di methyl K20, tri methyl K20)</td><td>Abcam</td><td>ab78517</td><td>Dilution factor- 1:500</td></tr><tr><td>Rabbit anti-Rec8</td><td>Courtesy of Dr. Karen Schindler</td><td>N/A</td><td>Dilution factor- 1:10,000</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Secondary Antibodies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Donkey anti-goat IgG (H+L) Alexa Fluor 568 conjugate</td><td>Thermo-Fisher Scientific</td><td>A-11057</td><td>Dilution factor- 1:500</td></tr><tr><td>Donkey anti-mouse IgG (H+L) Alexa Fluor 488 conjugate</td><td>Thermo-Fisher Scientific</td><td>A-21202</td><td>Dilution factor- 1:500</td></tr><tr><td>Donkey anti-rabbit IgG (H+L) Alexa Fluor 647 conjugate</td><td>Thermo-Fisher Scientific</td><td>A-31573</td><td>Dilution factor- 1:500</td></tr><tr><td>Goat anti-mouse IgG (H+L) Alexa Fluor 568 conjugate</td><td>Thermo-Fisher Scientific</td><td>A-11004</td><td>Dilution factor- 1:500</td></tr><tr><td>Goat anti-Human IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488</td><td>Thermo-Fisher Scientific</td><td>A-11013</td><td>Dilution factor- 1:500</td></tr><tr><td>Goat anti-rabbit IgG (H+L) Alexa Fluor 568 conjugate</td><td>Thermo-Fisher Scientific</td><td>A-11011</td><td>Dilution factor- 1:500</td></tr></tbody></table>

chromatin spread preparations for the analysis of mouse oocyte progression from prophase to metaphase ii

טכניקות התפשטות כרומטין היה בשימוש נרחב כדי להעריך לוקליזציה דינמי של חלבונים שונים במהלך gametogenesis, במיוחד עבור יצירת זרע. טכניקות אלה מאפשרות ויזואליזציה של חלבון ודפוסי לוקליזציה DNA במהלך אירועים meiotic כגון כרומוזום הומולוגי זיווג, synapsis, ה-DNA תיקון. בעוד כמה פרוטוקולים תוארו בספרות, כרומטין כללי התפשטות באמצעות oocytes prophase בתרבית של טכניקות הם מוגבלים וקשה בשל העיתוי של מיוזה חניכה של השחלות עוברית. לשם השוואה, ניתן לאסוף prophase spermatocytes של עכברים זכר לנוער עם תשואות גבוהות ללא צורך microdissection. עם זאת, קשה להשיג אוכלוסיה מסונכרן טהור של תאים בשלבים מסוימים עקב הטרוגניות של אוכלוסיות meiotic, פוסט-meiotic תא הנבט ב בזרמה לנוער ומבוגרים. בשלבים מאוחרים יותר של מיוזה, זה יתרון כדי להעריך oocytes עוברים מיוזה I (MI) או מיוזה II (MII), כי קבוצות של oocytes בוגר יכול להיות שנאסף עכברים נקבה בוגרת מגורה כדי לחדש את המיוזה בתרבות. . הנה, שיטות ההכנות כרומטין meiotic להתפשט באמצעות oocytes גזור מן העובר, neonatal, השחלות למבוגרים מתוארים בליווי הדגמות וידאו. כרומוזום missegregation םיעוריא oocytes יונקים הם בתדירות גבוהה, במיוחד במהלך MI. שיטות אלה ניתן להשתמש כדי להעריך ולאפיין את ההשפעות של מוטציות שונות או חשיפות סביבתיות בשלבים שונים של oogenesis. שכן, ישנם הבדלים ברורים בין oogenesis לבין יצירת זרע מהטכניקות שתוארו בתוך הם לא יסולא בפז כדי להגדיל את ההבנה שלנו של יונקים oogenesis ותכונות dimorphic מינית של דינמיקה כרומוזום וחלבון במהלך המיוזה .

תארנו שתי טכניקות בשביל להמחיש והערכה הכרומוזומים. oocytes meiotic. הטכניקה הראשונה היא ששירתה לקראת הערכת התקדמות prophase של השחלות עובריים ו neonatal. Prophase כרומטין התפשטות ההכנות בעל ערך רב להמחשת תהליכים דינאמיים רבים במהלך המיוזה, כולל כרומוזום זיווג, synapsis, desynapsis, רקומבינציה הומו?...

Watch this Scientific Journal Video about Chromatin Spread Preparations for the Analysis of Mouse Oocyte Progression from Prophase to Metaphase II at JoVE.com

הכנות לממרח כרומטין לאנליזה של התקדמות ביציות בעכבר מפרופאזה למטאפאזה II | גנטיקה | וִידֵאוֹ

Chromatin Spread Preparations for the Analysis of Mouse Oocyte Progression from Prophase to Metaphase II

כרומטין להפיץ את ההכנות לניתוח של העכבר Oocyte התקדמות מן Prophase כדי מפה של השני

Oogenesis ביונקים ידוע להיות מועדת לטעויות, במיוחד עקב כרומוזום missegregation. כתב יד זה מתאר שיטות הכנה כרומטין להפיץ prophase העכבר, מפה של אני, oocytes ביים השני. טכניקות יסוד אלה מאפשרים לימוד חלבוני כרומטין מכורך כרומוזום מורפולוגיה ברחבי oogenesis יונקים.

Chromatin spread techniques have been widely used to assess the dynamic localization of various proteins during gametogenesis, particularly for ...

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Unit 2

Meiosis

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15.3K Views.  Johns Hopkins University Bloomberg School of Public Health. Oogenesis ביונקים ידוע להיות מועדת לטעויות, במיוחד עקב כרומוזום missegregation. כתב יד זה מתאר שיטות הכנה כרומטין להפיץ prophase העכבר, מפה של אני, oocytes ביים השני. טכניקות יסוד אלה מאפשרים לימוד חלבוני כרומטין מכורך כרומוזום מורפולוגיה ברחבי oogenesis יונקים.

Video: Chromatin Spread Preparations for the Analysis of Mouse Oocyte Progression from Prophase to Metaphase II

Isolation and Characterization of Mouse Antral Oocytes Based on Nucleolar Chromatin Organization

This protocol describes a simple and quick method to isolate and characterize mouse antral GV (Germinal Vesicle) oocytes as able (SN, Surrounded Nucleolus) or unable (NSN, Not Surrounded Nucleolus) to develop to the blastocyst stage after in vitro maturation (IVM) and in vitro fertilization (IVF). It makes use of Hoeschst33342 (or any other DNA intercalating dye) able to bind to the heterochromatin of the nucleolus showing a ring in the SN oocytes or not, like in the NSN oocytes. This represents the easiest and quickest way to sort both antral oocytes that can be eventually used for IVM or IVF procedures. 
Briefly, the protocol consists of the following steps: hormone injection to stimulate follicular growth; isolation of the oocytes at the GV stage from the antral compartment by puncturing the ovary with a sterile needle; preparation of thin glass pipettes for mouth pipetting of the oocytes; sorting of the oocytes with Hoechst33342 prepared at a supravital concentration; IVM, IVF or any other molecular/cellular analysis.
Unfortunately there are still few evidences to sort SN and NSN oocytes using less invasive techniques. If and once they will be identified, they could be potentially applied to human assisted reproductive technologies, although with several aspects that should be modified. To date, this technique has potential implications to dramatically increase IVM and IVF successful procedures in both endangered and species with economic interest.

Here we present a protocol for the characterization of mouse antral oocytes based on nucleolar organization.

בידוד ואפיון של עכבר Antral ביציות בהתבסס על ארגון הכרומטין nucleolar

This protocol describes a simple and quick method to isolate and characterize mouse antral GV (Germinal Vesicle) oocytes as able (SN, Surrounded ...

Developmental Biology

Using Mouse Oocytes to Assess Human Gene Function During Meiosis I

Embryonic aneuploidy is the major genetic cause of infertility in humans. Most of these events originate during female meiosis, and albeit positively correlated with maternal age, age alone is not always predictive of the risk of generating an aneuploid embryo. Therefore, gene variants might account for incorrect chromosome segregation during oogenesis. Given that access to human oocytes is limited for research purposes, a series of assays were developed to study human gene function during meiosis I using mouse oocytes. First, messenger RNA (mRNA) of the gene and gene variant of interest are microinjected into prophase I-arrested mouse oocytes. After allowing time for expression, oocytes are synchronously released into meiotic maturation to complete meiosis I. By tagging the mRNA with a sequence of a fluorescent reporter, such as green fluorescent protein (Gfp), the localization of the human protein can be assessed in addition to the phenotypic alterations. For example, gain or loss of function can be investigated by establishing experimental conditions that challenge the gene product to fix meiotic errors. Although this system is advantageous in investigating human protein function during oogenesis, adequate interpretation of results should be undertaken given that protein expression is not at endogenous levels and, unless controlled for (i.e. knocked out or down), murine homologs are also present in the system.

As the genetic variants associated with human disease begin to become uncovered, it is becoming increasingly important to develop systems with which to rapidly evaluate the biological significance of those identified variants. This protocol describes methods for evaluating human gene function during female meiosis I using mouse oocytes.

באמצעות העכבר Oocytes כדי להעריך תפקוד הגן האנושי במהלך המיוזה אני

Embryonic aneuploidy is the major genetic cause of infertility in humans. Most of these events originate during female meiosis, and albeit positively ...

Analysis of Chromosome Segregation, Histone Acetylation, and Spindle Morphology in Horse Oocytes

The field of assisted reproduction has been developed to treat infertility in women, companion animals, and endangered species. In the horse, assisted reproduction also allows for the production of embryos from high performers without interrupting their sports career and contributes to an increase in the number of foals from mares of high genetic value. The present manuscript describes the procedures used for collecting immature and mature oocytes from horse ovaries using ovum pick-up (OPU). These oocytes were then used to investigate the incidence of aneuploidy by adapting a protocol previously developed in mice. Specifically, the chromosomes and the centromeres of metaphase II (MII) oocytes were fluorescently labeled and counted on sequential focal plans after confocal laser microscope scanning. This analysis revealed a higher incidence in the aneuploidy rate when immature oocytes were collected from the follicles and matured in vitro compared to in vivo. Immunostaining for tubulin and the acetylated form of histone four at specific lysine residues also revealed differences in the morphology of the meiotic spindle and in the global pattern of histone acetylation. Finally, the expression of mRNAs coding for histone deacetylases (HDACs) and acetyl-transferases (HATs) was investigated by reverse transcription and quantitative-PCR (q-PCR). No differences in the relative expression of transcripts were observed between in vitro and in vivo matured oocytes. In agreement with a general silencing of the transcriptional activity during oocyte maturation, the analysis of the total transcript amount can only reveal mRNA stability or degradation. Therefore, these findings indicate that other translational and post-translational regulations might be affected.
Overall, the present study describes an experimental approach to morphologically and biochemically characterize the horse oocyte, a cell type that is extremely challenging to study due to low sample availability. However, it can expand our knowledge on the reproductive biology and infertility in monovulatory species.

This manuscript describes an experimental approach to morphologically and biochemically characterize horse oocytes. Specifically, the present work illustrates how to collect immature and mature horse oocytes by ultrasound-guided ovum pick-up (OPU) and how to investigate chromosome segregation, spindle morphology, global histone acetylation, and mRNA expression.

ניתוח של הפרדת כרומוזום, אצטון היסטון, ומורפולוגיה ציר ב סוס ביציות

The field of assisted reproduction has been developed to treat infertility in women, companion animals, and endangered species. In the horse, assisted ...

Preparation of Meiotic Chromosome Spreads from Zebrafish Spermatocytes

Meiosis is the key cellular process required to create haploid gametes for sexual reproduction. Model organisms have been instrumental in understanding the chromosome events that take place during meiotic prophase, including the pairing, synapsis, and recombination events that ensure proper chromosome segregation. While the mouse has been an important model for understanding the molecular mechanisms underlying these processes, not all meiotic events in this system are analogous to human meiosis. We recently demonstrated the exciting potential of the zebrafish as a model of human spermatogenesis. Here we describe, in detail, our methods to visualize meiotic chromosomes and associated proteins in chromosome spread preparations. These preparations have the advantage of allowing high resolution analysis of chromosome structures. First, we describe the procedure for dissecting testes from adult zebrafish, followed by cell dissociation, lysis, and spreading of the chromosomes. Next, we describe the procedure for detecting the localization of meiotic chromosome proteins, by immunofluorescence detection, and nucleic acid sequences, by fluorescence in situ hybridization (FISH). These techniques comprise a useful set of tools for the cytological analysis of meiotic chromatin architecture in the zebrafish system. Researchers in the zebrafish community should be able to quickly master these techniques and incorporate them into their standard analyses of reproductive function.

Nuclear surface spreads are an indispensable tool for studying chromosome events during meiosis. Here we demonstrate a method to prepare and visualize meiotic chromosomes during prophase I from zebrafish spermatocytes.

הכנת כרומוזום מיוסטי כפולות מדגים בזימטציטים

Meiosis is the key cellular process required to create haploid gametes for sexual reproduction. Model organisms have been instrumental in understanding ...

Evaluation of the Spindle Assembly Checkpoint Integrity in Mouse Oocytes

Aneuploidy is the leading genetic abnormality causing early miscarriage and pregnancy failure in humans. Most errors in chromosome segregation that give rise to aneuploidy occur during meiosis in oocytes, but why oocyte meiosis is error-prone is still not fully understood. During cell division, cells prevent errors in chromosome segregation by activating the spindle assembly checkpoint (SAC). This control mechanism relies on detecting kinetochore (KT)-microtubule (MT) attachments and sensing tension generated by spindle fibers. When KTs are unattached, the SAC is activated and prevents cell-cycle progression. The SAC is activated first by MPS1 kinase, which triggers the recruitment and formation of the mitotic checkpoint complex (MCC), composed of MAD1, MAD2, BUB3, and BUBR1. Then, the MCC diffuses into the cytoplasm and sequesters CDC20, an anaphase-promoting complex/cyclosome (APC/C) activator. Once KTs become attached to microtubules and chromosomes are aligned at the metaphase plate, the SAC is silenced, CDC20 is released, and the APC/C is activated, triggering the degradation of Cyclin B and Securin, thereby allowing anaphase onset. Compared to somatic cells, the SAC in oocytes is not as effective because cells can undergo anaphase despite having unattached KTs. Understanding why the SAC is more permissive and if this permissiveness is one of the causes of chromosome segregation errors in oocytes still needs further investigation. The present protocol describes the three techniques to comprehensively evaluate SAC integrity in mouse oocytes. These techniques include using nocodazole to depolymerize MTs to evaluate the SAC response, tracking SAC silencing by following the kinetics of Securin destruction, and evaluating the recruitment of MAD2 to KTs by immunofluorescence. Together these techniques probe mechanisms needed to produce healthy eggs by providing a complete evaluation of SAC integrity.

Error in chromosome segregation is a common feature in oocytes. Therefore, studying the spindle assembly checkpoint gives important clues about the mechanisms needed to produce healthy eggs. The present protocol describes three complementary assays to evaluate spindle assembly checkpoint integrity in mouse oocytes.

הערכת שלמות מחסום הרכבת הציר בביציות עכבר

Aneuploidy is the leading genetic abnormality causing early miscarriage and pregnancy failure in humans. Most errors in chromosome segregation that give ...

Mouse Oocyte Microinjection, Maturation and Ploidy Assessment

Mistakes in chromosome segregation lead to aneuploid cells. In somatic cells, aneuploidy is associated with cancer but in gametes, aneuploidy leads to infertility, miscarriages or developmental disorders like Down syndrome. Haploid gametes form through species-specific developmental programs that are coupled to meiosis. The first meiotic division (MI) is unique to meiosis because sister chromatids remain attached while homologous chromosomes are segregated. For reasons not fully understood, this reductional division is prone to errors and is more commonly the source of aneuploidy than errors in meiosis II (MII) or than errors in male meiosis 1,2. 
In mammals, oocytes arrest at prophase of MI with a large, intact germinal vesicle (GV; nucleus) and only resume meiosis when they receive ovulatory cues. Once meiosis resumes, oocytes complete MI and undergo an asymmetric cell division, arresting again at metaphase of MII. Eggs will not complete MII until they are fertilized by sperm. Oocytes also can undergo meiotic maturation using established in vitro culture conditions 3. Because generation of transgenic and gene-targeted mouse mutants is costly and can take long periods of time, manipulation of female gametes in vitro is a more economical and time-saving strategy. 
Here, we describe methods to isolate prophase-arrested oocytes from mice and for microinjection. Any material of choice may be introduced into the oocyte, but because meiotically-competent oocytes are transcriptionally silent 4,5 cRNA, and not DNA, must be injected for ectopic expression studies. To assess ploidy, we describe our conditions for in vitro maturation of oocytes to MII eggs. Historically, chromosome-spreading techniques are used for counting chromosome number 6. This method is technically challenging and is limited to only identifying hyperploidies. Here, we describe a method to determine hypo-and hyperploidies using intact eggs 7-8. This method uses monastrol, a kinesin-5 inhibitor, that collapses the bipolar spindle into a monopolar spindle 9 thus separating chromosomes such that individual kinetochores can readily be detected and counted by using an anti-CREST autoimmune serum. Because this method is performed in intact eggs, chromosomes are not lost due to operator error.

Oocytes are prone to aneuploidy due to errors in chromosome segregation during meiotic maturation. Aneuploid eggs can cause infertility, miscarriages or developmental disorders like Down syndrome. Here, we describe methods to introduce materials of choice into oocytes and methods to study meiotic maturation and assess ploidy.

הביצית Microinjection עכבר, הבשלה ו Ploidy הערכה

Mistakes in chromosome segregation lead to aneuploid cells.  In somatic cells, aneuploidy is associated with cancer but in gametes, aneuploidy leads to ...

A Cell Free Assay to Study Chromatin Decondensation at the End of Mitosis

During the vertebrate cell cycle chromatin undergoes extensive structural and functional changes. Upon mitotic entry, it massively condenses into rod shaped chromosomes which are moved individually by the mitotic spindle apparatus. Mitotic chromatin condensation yields chromosomes compacted fifty-fold denser as in interphase. During exit from mitosis, chromosomes have to re-establish their functional interphase state, which is enclosed by a nuclear envelope and is competent for replication and transcription. The decondensation process is morphologically well described, but in molecular terms poorly understood: We lack knowledge about the underlying molecular events and to a large extent the factors involved as well as their regulation. We describe here a cell-free system that faithfully recapitulates chromatin decondensation in vitro, based on mitotic chromatin clusters purified from synchronized HeLa cells and X. laevis egg extract. Our cell-free system provides an important tool for further molecular characterization of chromatin decondensation and its co-ordination with processes simultaneously occurring during mitotic exit such as nuclear envelope and pore complex re-assembly.

The molecular mechanisms of the decondensation of highly compacted mitotic chromatin are ill-defined. We present a cell-free assay based on mitotic chromatin clusters isolated from HeLa cells and Xenopus laevis egg extract that faithfully reconstitutes the decondensation process in vitro.

תא Assay חינם לחקר הכרומטין Decondensation בסוף מיטוזה

During the vertebrate cell cycle chromatin undergoes extensive structural and functional changes. Upon mitotic entry, it massively condenses into rod ...

Meiotic Spindle Assessment in Mouse Oocytes by siRNA-mediated Silencing

Errors in chromosome segregation during meiotic division in gametes can lead to aneuploidy that is subsequently transmitted to the embryo upon fertilization. The resulting aneuploidy in developing embryos is recognized as a major cause of pregnancy loss and congenital birth defects such as Down&#8217;s syndrome. Accurate chromosome segregation is critically dependent on the formation of the microtubule spindle apparatus, yet this process remains poorly understood in mammalian oocytes. Intriguingly, meiotic spindle assembly differs from mitosis and is regulated, at least in part, by unique microtubule organizing centers (MTOCs). Assessment of MTOC-associated proteins can provide valuable insight into the regulatory mechanisms that govern meiotic spindle formation and organization. Here, we describe methods to isolate mouse oocytes and deplete MTOC-associated proteins using a siRNA-mediated approach to test function. In addition, we describe oocyte fixation and immunofluorescence analysis conditions to evaluate meiotic spindle formation and organization.

Here, we present a protocol for specific siRNA-mediated mRNA depletion followed by immunofluorescence analysis to evaluate meiotic spindle assembly and organization in mouse oocytes. This protocol is suitable for in vitro depletion of transcripts and functional assessment of different spindle and/or MTOC-associated factors in oocytes.

Meiotic ציר ההערכה בעכבר ביציות על ידי השתקה בתיווך siRNA

Errors in chromosome segregation during meiotic division in gametes can lead to aneuploidy that is subsequently transmitted to the embryo upon ...

Techniques for Imaging Prometaphase and Metaphase of Meiosis I in Fixed Drosophila Oocytes

Chromosome segregation in human oocytes is error prone, resulting in aneuploidy, which is the leading genetic cause of miscarriage and birth defects. The study of chromosome behavior in oocytes from model organisms holds much promise to uncover the molecular basis of the susceptibility of human oocytes to aneuploidy. Drosophila melanogaster is amenable to genetic manipulation, with over 100 years of research, community, and technique development. Visualizing chromosome behavior and spindle assembly in Drosophila oocytes has particular challenges, however, due primarily to the presence of membranes surrounding the oocyte that are impenetrable to antibodies. We describe here protocols for the collection, preparation, and imaging of meiosis I spindle assembly and chromosome behavior in Drosophila oocytes, which allow the molecular dissection of chromosome segregation in this important model organism.

Techniques for Imaging Prometaphase and Metaphase of Meiosis I in Fixed Drosophila Oocytes

We present protocols for the collection, preparation, and imaging of mature Drosophila oocytes. These methods allow the visualization of chromosome behavior and spindle assembly and function during meiosis.

טכניקות הדמיה prometaphase ו Metaphase של המיוזה ואני קבוע<em&gt; תסיסנית</em&gt; ביציות

Chromosome segregation in human oocytes is error prone, resulting in aneuploidy, which is the leading genetic cause of miscarriage and birth defects. The ...

Preparation of Meiotic Chromosome Spreads from Mouse Spermatocytes

Mammalian meiosis is a dynamic developmental process that occurs in germ cells and can be studied and characterized. Using a method to spread nuclei on the surface of slides (rather than dropping them from a height), we demonstrate an optimized technique on mouse spermatocytes that was first described in 1997. This method is widely used in laboratories to study mammalian meiosis because it yields a plethora of high quality nuclei undergoing substages of prophase I. Seminiferous tubules are first placed in a hypotonic solution to swell spermatocytes. Then spermatocytes are released into a sucrose solution to create a cell suspension, and nuclei are spread onto fixative-soaked glass slides. Following immunostaining, a diversity of proteins germane to meiotic processes can be examined. For example, proteins of the synaptonemal complex, a tripartite structure that connects the chromosome axes/cores of homologs together can be easily visualized. Meiotic recombination proteins, which are involved in repair of DNA double-strand breaks by homologous recombination, can also be immunostained to evaluate progression of prophase I. Here we describe and demonstrate in detail a technique widely used to study mammalian meiosis in spermatocytes from juvenile or adult male mice.

Meiosis is the developmental process by which gametes are formed through a single round of DNA replication and two successive rounds of chromosome segregation. Mammalian meiosis can be examined by utilizing a technique to prepare meiotic chromosome spreads. Here, we demonstrate a method of preparing surface-spread nuclei from mouse spermatocytes.

הכנה של כרומוזום Meiotic מתפשטת העכבר Spermatocytes

Mammalian meiosis is a dynamic developmental process that occurs in germ cells and can be studied and characterized. Using a method to spread nuclei on ...