Preparation of Meiotic Chromosome Spreads from Mouse Oocytes for Assessment of Synapsis and Recombination

Summary

Figure 1: End-to-end framework. The figure illustrates end-to-end framework for multimodal knowledge Graph VISHAM-KG. Please click here to view a larger version of this figure.

Abstract

Three critical and interdependent processes define meiotic prophase I: homologous chromosomes must pair together, a proteinaceous structure called the synaptonemal complex forms to tether homologs together (synapsis), and homologs undergo recombination, reciprocally exchanging genetic material to form crossovers (COs). Errors in these processes can result in premature ovarian insufficiency, aneuploidy, and ultimately, pregnancy loss and infertility. Meiotic recombination is particularly error-prone in oocytes, with over 7% of human oocytes containing at least one chromosome pair without a crossover, and between 20%-80% of eggs versus 2.5%-7% of sperm are aneuploid. However, it remains unclear why chromosomal errors show such striking sex disparities. The present protocol describes methods for the preparation and analysis of oocyte prophase I and metaphase I chromosome spreads from fetal and juvenile mice, respectively. To trace critical chromosome dynamics throughout meiotic prophase I, this protocol employs immunofluorescence staining of common markers for meiotic recombination (RAD51 for double strand breaks, MSH4 for CO intermediates, and MLH1/MLH3 to identify most COs) and synapsis (SYCP3 for chromosome axes, SYCP1 for synapsed regions, and HORMAD1 for asynapsed regions). Further, this protocol uses in vitro maturation of oocytes collected to assess the number of paired chromosomes (bivalents) and the total number of crossovers (chiasmata) in metaphase I. Together, these techniques provide a comprehensive and quantitative framework to examine mechanisms regulating early chromosome dynamics in female meiosis.

Introduction

Meiosis is a specialized cell division essential to the production of haploid gametes. During prophase I, homologous chromosomes (each consisting of sister chromatids held together by cohesin) must accomplish three critical tasks: 1) recognize and pair with each other, 2) zip together by forming the synaptonemal complex (SC) between their lateral axes, and 3) exchange DNA via homologous recombination, resulting in crossovers (COs) between non-sister chromatids^1,2. The physical connection resulting from crossover formation, maintained by cohesin, ensures proper chromosome biorientation and segregation during me....

Protocol

All animal handling and procedures were performed following approval by the Cornell Institutional Animal Care and Use Committee (IACUC) under protocol 2004-0063.

1. Preparation of oocyte prophase I chromosome spread slide

1. In late afternoon, set up timed matings of C57BL/6J female mice (2 - 6 months old) with males (2 - 12 months old). Beginning the next day, check females each morning (before 9 AM) for a copulatory plug. Noon on the day of the plug is defined as 0.5 dpc.
2. Prepare the following prior to sacrificing pregnant mice.
   1. Set up a humidified chamber with room temperature H₂....

Results

Evaluation of synapsis defects in pachytene-like cells
This experiment evaluates synapsis defects in pachytene oocytes and monitors prophase I progression (Figure 3A). Immunostaining for SYCP3 (axial/lateral SC elements), SYCP1 (central SC element), and HORMAD1 (unsynapsed/desynapsed chromosome axes) enables staging and synapsis assessment. HORMAD1 colocalizes with SYCP3 during zygonema and diplonema (before synapsis and after desynapsis).......

Discussion

Mammalian oocytes are particularly error-prone compared to spermatocytes. While the oocyte's less efficient spindle assembly checkpoint (SAC) contributes to this vulnerability^14,52,53, Hassold and Hunt propose that multiple hits lead to aneuploidy, with defective recombination being a primary factor⁵⁴. Human studies link aneuploidy to insufficient crossover assurance and suboptimal crossover positioni.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.45 μm Syringe Filter	Millipore Sigma	SLHAR33SB	for making mouth-operated pipette
0.5 M EDTA, pH 8.0	Corning	46-034-CI	for HEB
1 mL Sterile Syringe with 25-gauge, 16 mm needle	Fisher Scientific	14-817-125	for rupturing ovaries
100 x 15 mm Petri dish	VWR	25384-302
14.6 cm disposable borosilicate glass Pasteur pipets	Fisher Scientific	13-678-20B	for making mouth-operated pipette
35 x 10 mm Petri dish	Corning	351008
40 mm x 0.45 mm #3 Stainless Steel Pins for Entomology	Amazon	B0CSSPW5S7	for pinning fetal mice for dissection
Acetic Acid, Glacial	Fisher Scientific	A38-212	for Carnoy's fixative
Aspirator tube assemblies for calibrated microcapillary pipettes	Millipore Sigma	A5177-5EA	for making mouth-operated pipette
B.P.I. Watch Glass	NDS Technologies	1580-00	Alternatively use a 30 mm dia x 12 mm deep embryo dish (Electron Microscopy Sciences SKU: 70543-30)
Boric Acid	Fisher Scientific	BP168-1	for adjusting pH of HEB, PFA
Bovine Serum Albumin	Millipore Sigma	A7906-100G	for ADB
CO2 Tank			for incubator
Corning Microscope Slides	Millipore Sigma	CLS294875X25	L x W 75 mm x 25 mm, frosted one side
Corning microscope slides, frosted one side, one end	VWR	294875X25
Cover glass rectangular. 60 x 24 mm, #1.5	VWR	48393-251
Dissecting Dish	Electron Microscopy Sciences	70540
Dissection Microscope			Any standard model
Dissection Scissors, Sharp Tip, 4.5"	VWR	82027-578
Extra fine, sharp tip forceps	VWR	76548-836
Giemsa Stain	Millipore Sigma	GS500-500	for staining chiasmata spreads
ImmEdge Hydrophobic Barrier PAP Pen	Vector Laboratories	H-4000
Incubator			Any standard model with CO2 and water jacketed technology
IVF Workstation	K-Systems		Any model with a heated benchtop
Kodak Photo-Flo 200 Solution	BH Photo & Video	1464510	for slide washes
Methanol	Fisher Scientific	A412-4	for Carnoy's fixative
MP Biomedicals Coplin Staining Jar	Fisher Scientific	ICN17006201
Normal Goat Serum	Gibco	16210-072	for ADB
Paraformaldehyde, EM Grade, Purified Prill	Electron Microscopy Sciences	19200
Permount	Fisher Scientific	SP15-100	for  mounting slides for chiasmata spreads
ProLong Glass Antifade Mountant with NucBlue Stain	Thermo Scientific	P36985	for mounting immunostained slides
Silicone tube, interior diameter: 0.125", exterior diameter: 0.25"	VWR	89068-474	for making mouth-operated pipette
Sodium Citrate Dihydrate	Fisher Scientific	S279-500	for HEB, hypotonic solution
StainTray Slide Staining System	Electron Microscopy Sciences	71396-B	For use as a humidified chamber. Must use the black lid for immunofluorescence staining. May alternatively line the bottom of an opaque slide box or tupper ware with damp paper towels for use as a humidified chamber
Stripper Tips - 100	Cooper Surgical	MXL3-100	for mouth-operated pipette
Stripper Tips - 150	Cooper Surgical	MXL3-150	for mouth-operated pipette
Sucrose	Millipore Sigma	S-8501	for HEB, Sucrose hypotonic solution
Tris Base	Fisher Scientific	BP152-5	for HEB
Triton X-100	Fisher Scientific	BP151-100	for PFA, ADB, and Triton-PBS
United Scientific Coplin Staining Jar	Fisher Scientific	S17495A	opaque white plastic for washes and blocking slides after addition of photosensitive secondary antibodies
United Scientific Glass Concavity Slides	Fisher Scientific	S139533	1.3 mm thick, 2 well count
Vannas Microdissection Scissors 5mm straight	VWR	76457-358
Zeiss Axio Imager.Z2, Colibri 7	Carl Zeiss	000000-2624-307
Zen Blue 3.11 Software	Carl Zeiss		Image acquisition and analysis software
Antibodies
Alexa Fluor 488 AffiniPure F(ab')? Fragment Goat Anti-Mouse IgG, Fc fragment specific	Jackson ImmunoResearch Laboratories Inc.	115-546-071	used 1:1,000 dilution
Alexa Fluor 488 AffiniPure F(ab')? Fragment Goat Anti-Rabbit IgG, Fc fragment specific	Jackson ImmunoResearch Laboratories Inc.	111-546-046	used 1:1,000 dilution
Alexa Fluor 647 AffiniPure F(ab')? Fragment Goat Anti-Human IgG, Fcγ fragment specific	Jackson ImmunoResearch Laboratories Inc.	109-606-170	used 1:1,000 dilution
Alexa Fluor 647 AffiniPure Goat Anti-Guinea Pig IgG, Fc fragment specifi	Jackson ImmunoResearch Laboratories Inc.	106-605-008	used 1:1,000 dilution
Guinea Pig anti-MLH3 pAb			custom made with Thermo-Fisher, used 1:500 dilution
Guinea Pig anti-SYCP1 pAb			from Christer Höög (Kouznetsova et al. 2005), used 1:200 dilution
Human anti-Centromere protein pAb	Antibodies Inc.	15-234	used 1:1,000 dilution
Mouse anti-MLH1 mAb	BD Biosciences	550838	used 1:100 dilution
Mouse anti-SYCP3 mAb	Abcam	ab97672	used 1:1,000 dilution
Rabbit anti-HORMAD1 pAb	Proteintech	13917-1-AP	used 1:500 dilution
Rabbit anti-MSH4 pAb	Abclonal	A8556	used 1:100 dilution
Rabbit anti-RAD51 pAb	Millipore	PC130	used 1:500 dilution
Rabbit anti-SYCP3 pAb	Abcam	ab15093	used 1:1,000 dilution
Rhodamine Red-X (RRX) AffiniPure F(ab')? Fragment Goat Anti-Mouse IgG, Fc fragment specific	Jackson ImmunoResearch Laboratories Inc.	115-296-071	used 1:1,000 dilution
Rhodamine Red-X (RRX) AffiniPure F(ab')? Fragment Goat Anti-Rabbit IgG, Fc fragment specific	Jackson ImmunoResearch Laboratories Inc.	111-296-046	used 1:1,000 dilution