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

Summary

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.

Abstract

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.

Introduction

Meiosis is a complex cellular event in which a single round of DNA replication is followed by two successive rounds of cell division. Several meiosis-specific events must be coordinated during the initial stages of meiosis to ensure accurate chromosome segregation. These events include the completion of homologous recombination, co-orientation of sister kinetochores during the first meiotic division, and the stepwise loss of cohesin complexes to resolve chiasmata between homologs. Precise regulation of these processes is necessary to maintain fertility and to prevent chromosome missegregation events that can lead to genetic developmental disorders and spontaneous misc....

Protocol

The use of mice was approved by the Institutional Animal Care and Use Committee of Johns Hopkins University. Experiments were performed on juvenile (20 - 26 days post-partum, dpp) C57BL/6J mice, taking advantage of the semi-synchronous first wave of spermatogenesis. However, this technique can also be performed using adult mice.

1. Dissection and isolation of mouse seminiferous tubules

1. Prepare the fix/lysis solution and antibody dilution buffer (ADB) described in Table 1 and Table 2.
2. Prepare one 35-mm Petri dish with 3 mL of 1x phosphate buffered saline (1x PBS, pH 7.4), and another 35....

Results

Here, we have used the tubule squash method to visualize transitory cell populations undergoing the prophase to metaphase I (G2/MI) transition, which were enriched by harvesting testes from juvenile wild-type mice undergoing the first wave of spermatogenesis (24 dpp). Figure 1 depicts representative images of the various cell stages that can be visualized using the tubule squash method. Enriched populations of metaphase I spermatocytes were visualized using a.......

Discussion

Mice have proven to be a useful model organism for studying the cellular events that govern meiotic progression during spermatogenesis. Further, it is necessary to develop tools catered to the study of spermatogenesis because many events, such as exit from meiotic prophase I, are sexually dimorphic. This protocol describes a seminiferous tubule squash method for visualization and study of different stages of mouse spermatogenesis. This method preserves cellular integrity and thereby allows detailed analyses of nuclear an.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
16% Paraformaldehyde Aqueous	Electron Microscopy Sciences (EMS)	15710
10x PBS	Quality Biological	119-069-161
Triton X-100	Sigma	T8787
BSA	Sigma	A1470
Horse Serum	Sigma	H-1270
35mm x 10mm Petri Dish, Sterile, non-treated	CellTreat	P886-229638
Poly-L-lysine coated glass slides	Sigma	P0425-72EA
Liquid Blocker Pen	Electron Microscopy Sciences (EMS)	71310
Humid Box	Evergreen	240-9020-Z10
Wheaton Coplin Glass Staining Dish for 5 or 10 Slides	Fisher	08-813E
VECTASHIELD Antifade Mounting Medium with DAPI	Vector Labs	H-1200
Microscope Cover Slides (22mmx60mm)	Fisher	12-544-G
Clear Nail Polish	Amazon	N/A
Microsopes
Name	Company	Catalog Number	Comments
SteREO Discovery.V8	Zeiss	495015-0001-000
Observer Z1	Zeiss	4109431007994000
Zeiss ZEN 2012 blue edition image software	Zeiss
ORCA-Flash 4.0 CMOS camera	Hamamatsu
Primary Antibodies
Name	Company	Catalog Number	Comments
Mouse anti-SYCP3	Santa Cruz	sc-74569	1 in 50
Rabbit anti-SYCP3	Fisher (Novus)	NB300-231	1 in 1000
Goat anti-SCP3	Santa Cruz	sc-20845	1 in 50
Human anti-Centromere Protein	Antibodies Incorporated	15-235	1 in 100
Mouse anti-alpha tubulin	Sigma	T9026	1 in 1000
Mouse anti-AIM1	BD Biosciences	611082	1 in 200
Mouse anti-γH2AX	Thermo Fisher	MA1-2022	1 in 500
Mouse anti-CENT3	Abnova	H00001070-M01	1 in 200
Rabbit anti-pericentrin	Abcam	ab4448	1 in 200
Rabbit anti-REC8	Courtesy of  Dr. Karen Schindler	N/A	1 in 1000