Preparation of Meiotic Chromosome Spreads from Mouse Spermatocytes

Summary

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.

Abstract

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.

Introduction

Mice are used extensively as a model organism to study meiosis in mammals. Both the normal developmental processes and defects that occur during meiosis can be evaluated. The timeline and progression of salient features that occur during prophase I and substages, as well as a multitude of specific proteins involved in processes crucial to regulation of meiosis can be characterized in both wild type and mutant mice. Several specialized processes that occur during meiosis I can be studied in detail (reviewed in Handel and Schimenti¹). These include DNA double-strand break (DSB) formation and repair, recombination, synaptonemal complex formation, ....

Protocol

All methods involving mice utilized high ethical and welfare standards, and were approved by the Institutional Animal Care and Use Committee at Drexel University College of Medicine.

1. Preparation of solutions and instruments needed

1. Prepare Hypotonic Extraction Buffer (HEB) in 50 mL of ultrapure laboratory grade water, pH 8.2 - 8.4 (Table 1). Make the solution fresh each time, keep on ice, and use within 2 h of preparing spreads; this is necessary to optimize the reducing and protease inhibiting activities of DTT and PMSF, respectively. Use 10 mL of HEB for each pair of mouse testes.
2. Prepare a 100 mM sucrose solution ....

Results

The ability of this method to provide large numbers of surface spread nuclei containing intact homologs depends on three main factors: 1) appropriate incubation time of seminiferous tubules in hypotonic buffer (i.e. HEB) to obtain adequately swelled spermatocyte nuclei that burst but do not disintegrate from prolonged incubation in HEB, 2) micropipetting the sucrose droplets of cells to obtain a separated suspension of nuclei that are not clumped together, and 3) tilting each fixative coa.......

Discussion

To obtain high numbers of well spread, good quality spermatocyte nuclei, the most critical steps of this protocol include appropriate incubation time of seminiferous tubules in HEB, appropriate mincing of seminiferous tubules, and the technique employed to spread the sucrose cell suspension across the fixative coated slide(s). We incubate seminiferous tubules from testes of wild type males ranging from postnatal day 15 to adulthood in HEB for 45 min, while testes from comparably aged Chtf18-null mice are incubat.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Photo-Flo 200	Kodak	1464510
16% Paraformaldehyde (formaldehyde aqueous solution)	Electron Microscopy Sciences	RT 15710	Dilute 16% solution into 4% working stocks with 1X PBS and freeze aliquots at -20 °C
Teflon printed 3 ring slides	Electron Microscopy Sciences	63418-11
Premium uncharged frosted end glass slides	Several commercial brands		Clean slides in ethyl or isopropyl alcohol and allow to drip dry prior to use; label slides on frosted end with a permanent marker or pencil depending on subsequent use of slides
Surgical scissors (sharp and blunt tips)	Fine Science Tools	14001-12	Different brand of a similar type will work
Fine tip surgical scissors (sharp tips)	Fine Science Tools	14058-11	Different brand of a similar type will work
Straight fine tip forceps	Fine Science Tools	11050-10	Different brand of a similar type will work
Curved medium tip forceps	Fisher	16100110	Different brand of a similar type will work
Scalpel handle	Fine Science Tools	10003-12	Different brand of a similar type will work
Mouse anti-SYCP3	Abcam	ab97672	Use at 1:300
Anti-centromere protein (derived from human CREST patient serum)	Antibodies Incorporated	15-234-0001	Use at 1:50
Humidified chamber			We use Nunc square culture dishes 500 cm2/well with moistened paper towels
Widefield microscope with epifluorescence	Leica microsystems		Any standard model
Coplin jars	Several commercial brands		50 ml capacity; 40 mm (1.6 in.) diameter, holds 10 slides (back to back)
Petri dishes	Several commercial brands		100 x 15 mm diameter, polystyrene sterile untreated