Isolation of Sertoli Cells and Peritubular Cells from Rat Testes

Summary

Primary Sertoli cells are required for studying testis-immune privilege, signal transduction during inflammation or infection, and utilization of their immunoprotective properties. Here we describe an enzyme-based protocol for the isolation of highly purified primary Sertoli cells and peritubular cells from rat testes.

Abstract

The testis, and in particular the male gamete, challenges the immune system in a unique way because differentiated sperm first appear at the time of puberty - more than ten years after the establishment of systemic immune tolerance. Spermatogenic cells express a number of proteins that may be seen as non-self by the immune system. The testis must then be able to establish tolerance to these neo-antigens on the one hand but still be able to protect itself from infections and tumor development on the other hand. Therefore the testis is one of a few immune privileged sites in the body that tolerate foreign antigens without evoking a detrimental inflammatory immune response. Sertoli cells play a key role for the maintenance of this immune privileged environment of the testis and also prolong survival of cotransplanted cells in a foreign environment. Therefore primary Sertoli cells are an important tool for studying the immune privilege of the testis that cannot be easily replaced by established cell lines or other cellular models. Here we present a detailed and comprehensive protocol for the isolation of Sertoli cells - and peritubular cells if desired - from rat testes within a single day.

Introduction

Testes produce male gametes and sexual hormones, i.e., androgens. The organ is composed of two compartments. In the interstitial compartment, that represents about 10-12% of total testicular volume¹, steroidogenesis takes place within the Leydig cells. The tubular compartment represents about 60-80% of the testicular volume¹ and contains germ cells and two types of somatic cells - peritubular cells and Sertoli cells. The testis is divided by connective tissue septa into 250-300 lobules, each containing 1-3 highly convoluted seminiferous tubules. These tubules are enclosed by a basal membrane, a sheet of collagen, and a circumferential la....

Protocol

1. Animal Ethics Statement

Experiments described here were performed according to the guidelines of the Regierungspräsidium Giessen, Germany, as the local authority and confirm to the German Code of Practice for the Care and Use of Animals for Experimental Purposes (Permission no: GI 20/23 Nr. A 31/2012).

2. Preparation of Media, Enzyme Solutions and Animals

1. Prepare 1% iodine alcohol by dissolving 1 g of iodine in 100 ml ethanol.
2. Prepare 2x 500 ml Dulbecco´s phosphate buffered saline (PBS) without Ca²⁺ and Mg²⁺ supplemented each with 7.5 ml D-glucose (100 g/L) and 5 ml 100x Penicill....

Results

The described procedure allows isolation of approximately 12 x 10⁷ Sertoli cells from 10 rat testes. 3 x 10⁶ cells are plated per well on a 6-well plate so that six to seven 6-well plates are available for experiments on day 7. The Trypsin-DNase I digestion is the most critical step during Sertoli cell isolation. If digestion at this point is advancing too far, the ratio of germ cells/Sertoli cells will disproportionally increase up to the end. During day 3-6 of cult.......

Discussion

The seminiferous tubules are bounded by a circular layer of peritubular cells and a basal lamina on the luminal side. Sertoli cells are resting on the basal lamina, establish the blood testis barrier through formation of occluding junctions between adjacent cells and provide the structural framework for the organisation of the seminiferous epithelium. Sertoli cells and adjacent spermatogenic cells maintain intimate contacts throughout germ cell development providing physical contact and communication alike. Therefore, wh.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Actin (smooth muscle) antibody clone 1A4	Dako	M0851	Monoclonal mouse anti human antibody. Use 1:100 dilution for immunofluorescence.
Albumin bovine fraction V Standard grade, lyophilized	Serva	11930.04	Filter (0.45 µm) BSA solutions used for immunofluorescence.
Corning Cell Strainers 70 µm	Corning	431751	White color
Collagenase A from Clostridium histolyticum	Roche	10103586001
DAPI mountant ProLong Gold Antifade	Life technologies	P-36931
D-glucose	Sigma	G8644	100 g/L
Dulbecco´s PBS without Ca2+/Mg2+	Gibco	14190-094
DNase I	Roche	10104159001
Electron microscope	Zeiss	EM 109S
Fluorescence microscope	Zeiss	Axioplan 2
Hyaluronidase from bovine testis	Sigma	H3506
Inverted light microscope	Olympus	CKX41	Routine cell culture microscope
Mouse IgG / IgM (H+L) polyclonal secondary Antibody	Life technologies	A-10684	Alexa Fluor 488 conjugate
Oil Red O	Sigma	O0625	Has replaced Sudan III and Sudan IV because of brighter color.
Paraformaldehyde	Sigma	P6148
Penicillin (5,000 U/ml)/ Streptomycin (5,000 µg/ml)	Gibco	15070-063	100x solution
RPMI-1640	Gibco	21875-034	Contains 300 mg/L L-glutamine
Trypsin from porcine pancreas	Sigma	T5266
Trypan Blue Stain (0.4%)	Gibco	15250-061
Trypsin inhibitor from soybean	Sigma	T6522	The Sigma product is considerably cheaper than the previously used BPTI (Aprotinin) from Roche.
Vimentin antibody (V9)	Sigma	V6630	Monoclonal mouse anti pig vimentin antibody.  Use 1:100 dilution for immunofluorescence.
Wistar WU rats	Charles River		Should be 19 days old on day of experiment.