Organ Culture and Whole Mount Immunofluorescence Staining of Mouse Wolffian Ducts

Summary

We present a method for isolation and culture of the mouse Wolffian duct (WD). We also demonstrate a detailed procedure for whole mount immunostaining of cultured/freshly isolated WDs with fluorescently tagged antibodies. Together, these techniques enable the study of WD development, coiling, and differentiation.

Abstract

Tubal morphogenesis is a fundamental requirement for the development of most mammalian organs, including the male reproductive system. The epididymis, an integral part of the male reproductive tract, is responsible for sperm storage, maturation, and transport. The adult epididymis is a highly coiled tube that develops from a simple and straight embryonic precursor known as Wolffian duct (WD). Proper coiling of the epididymis is essential for male fertility, as sperm in the testis are unable to fertilize an oocyte. However, the mechanism responsible for epididymal development and coiling remains unclear, partially due to the lack of whole organ culture and imaging methods. In this study, we describe an in vitro culture system and whole mount immunofluorescence protocol to better visualize the process of WD coiling and development, which may also be applied to study other tubular organs.

Introduction

The male reproductive system primarily consists of testis, the site for germ cell development and differentiation, and a complex ductal system that is required for the maturation, transport, and storage of sperm. Epididymis is a tubular organ that connects testis with vas deferens and mainly involved in post-testicular development and maturation of germ cells ¹. The highly coiled adult mouse epididymis develops from a simple and straight precursor tube, Wolffian duct (WD) ¹. The complex and coiled structure of epididymis is essential for sperm to acquire the capacity to fertilize female germ cells ². How such an essential organ for mal....

Protocol

Animal care and experimental procedures were conducted in accordance with the guidelines of the Animal Care and Ethics Committee of the University of Newcastle and confirmed to the New South Wales Animal Research Act, New South Wales Animal Research Regulation, and the Australian code for the care and use of animals for scientific purposes. All the procedures undertaken on mice were approved by the Animal Care and Ethics Committee of the University of Newcastle.

1. Time Mating

1. Pair 6 - 8 weeks old male and female mice just prior to the end of daylight cycle.
2. Check the females for the presence of vaginal plugs early mor....

Results

During development, the WD undergoes significant changes wherein a simple and straight tube is transformed into a highly complex and coiled duct. Using methods described above, WDs undergo a similar transformation in culture conditions. Here we have shown the results from WDs cultured for 3 d (Figure 3). To dissect molecular mechanisms involved in WD morphogenesis, the culture medium can be supplemented with inhibitors and activators targeting different signaling pathways. Figure 3C show.......

Discussion

The organ culture system has many advantages over the traditional cell culture system. This system retains the original structural relationships and interactions between different cell types. It mimics in vivo systems and gives more accurate information than cell culture studies where the niche factor is absent. The use of organ culture systems even have advantages over using the whole animal. These include the higher cost of using the whole animal, easy care and maintenance of organ culture system, etc.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
24-well plates	Corning, USA	3524	can be purchased from other vendors
Hank’s Balanced Salt Solution (HBSS)	GE Healthcare Life Sciences, USA	SH30031.02	can be purchased from other vendors
Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12)	Sigma, USA	D8437	Warm in 37 °C water bath before use
Fetal Bovine Serum (FBS)	Interpath,  Australia	SH30034.02	10% FBS used in culture medium
L-Glutamine	GE Healthcare Life Sciences, USA	SH30034.01	1% concentration used
Penicillin/Streptomycin	Gibco, USA	15070-063	1% concentration used
Whatman Nuclepore Polycarbonate Track-Etch	Whatman, USA	110409	Membrane (0.8 µm)
IWR1	Sigma, USA	10161-5mg	100 µM concentration used
Paraformaldehyde (PFA)	Electron Microscopy Sciences, USA	15710	16% stock, 4% in PBS for use. TOXIC - wear gloves and cannot be disposed of in the sink
Ethanol	Thermo Scientific Fisher, USA	214-20L PL	Absolute, make 25, 50 and 75% with milliQ water.
Tween-20	Thermo Scientific Fisher, USA	2509-500ml	Very viscous, careful while dispensing
Triton X-100	Sigma, USA	T8787-50ml	Very viscous, careful while dispensing
Di-Sodium Hydrogen Phosphate	Thermo Scientific Fisher, USA	621-500mg	can be purchased from other vendors
Sodium Di-hydrogen Phosphate	Ajax Finechem, USA	4745-500g	can be purchased from other vendors
Sodium Chloride	Thermo Scientific Fisher, USA	465-500g	can be purchased from other vendors
Cytokeratin8/Troma I	Developmental Studies Hybridoma Bank, USA (DHSB)	TROMA-I	1:250 in blocking buffer
active βcatenin	Cell Signalling Technology,  USA	D13A1	1:200 in blocking buffer
phospho-Histone 3	Millipore, MA, USA	06-570	1:200 in blocking buffer
Alexa488 Goat anti-rabbit IgG	Jackson ImmunoResearchLabs,  USA	111-545-047	1:250 in blocking buffer
Alexa594 Goat anti-Rat IgG	Jackson ImmunoResearchLabs,  USA	112-585-072	1:250 in blocking buffer
Glycerol	Thermo Fisher Scientific, USA	242-500ml	can be purchased from other vendors
n-Propyl galate	Sigma, USA	2370	-
2-(4-amidinophenyl)-1H -indole-6-carboxamidine (DAPI)	Sigma, USA	D9564-10mg	Use to stain nucleic acids (DNA)
Dimethyl sulfoxide (DMSO)	Thermo Fisher Scientific, USA	2225-500ml	Solvent
Cover Slips (24 x 50 mm)	Lomb Scientific	CS24501GP	-