An In Vivo Method to Study Mouse Blood-Testis Barrier Integrity

Summary

Here, we present a protocol to assess the blood-testis barrier integrity by injecting inulin-FITC into testes. This is an efficient in vivo method to study blood-testis barrier integrity that can be compromised by genetic and environmental elements.

Abstract

Spermatogenesis is the development of spermatogonia into mature spermatozoa in the seminiferous tubules of the testis. This process is supported by Sertoli cell junctions at the blood-testis barrier (BTB), which is the tightest tissue barrier in the mammalian body and segregates the seminiferous epithelium into two compartments, a basal and an adluminal. The BTB creates a unique microenvironment for germ cells in meiosis I/II and for the development of postmeiotic spermatids into spermatozoa via spermiogenesis. Here, we describe a reliable assay to monitor BTB integrity of mouse testis in vivo. An intact BTB blocks the diffusion of FITC-conjugated inulin from the basal to the apical compartment of the seminiferous tubules. This technique is suitable for studying gene candidates, viruses, or environmental toxicants that may affect BTB function or integrity, with an easy procedure and a minimal requirement of surgical skills compared to alternative methods.

Introduction

Mammalian spermatogenesis is considered a highly structured process that encompasses spermatogonial self-renewal and differentiation through spermatocytes into haploid spermatozoa via mitosis, meiosis, and spermiogenesis, during which dramatic biochemical and morphological changes occur. Developing germ cells are progressively transported from the base of the seminiferous tubule toward the lumen. This process is regulated by cell-cell contacts between germ cells and Sertoli cells^1,2. Adjacent Sertoli cells form the BTB that is located near the base of the seminiferous tubule. The BTB physically divides the epi....

Protocol

All performed animal experiments have been approved by the Nanjing Medical University committee. Male C57BL/6 mice were kept under controlled photoperiod conditions and were supplied with food and water.

1. Preparations

1. Microinjection capillaries
   1. Use microinjection capillaries with an outer diameter, inner dimeter, and length of 1.0 mm, 0.8 mm, and 10.0 cm, respectively.
   2. Pull glass capillaries with a capillary puller (Figure 1A). Test and adjust the settings depending on the capillary puller machine that is being used.
   3. Break the pipette tips with fo....

Results

The experimental set-up for performing the BTB integrity assay is shown in Figure 1. Pull and sharpen microinjection capillaries with a capillary puller and micropipette beveler, respectively (Figure 1A and 1C). The thermostatic heater and equipment for microinjection are illustrated in Figure 1B and 1D.

Discussion

Spermatogenesis takes place in the seminiferous epithelium and is a highly ordered and dynamic process that is governed by germ cells and somatic cells (e.g., Sertoli cells)¹³. The BTB structure, which is constructed by Sertoli cells, divides the seminiferous epithelium into a basal and an apical compartment. The development of meiotic and haploid germ cells occurs in the apical compartment which forms an immunological barrier¹⁴.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Capillary puller	SUTTER INSTRUMENT (USA)	P-97
10x PBS	Hyclone (USA)	SH30258.01	dilution to 1× in ddH2O
4’,6-diamidino-2-phenylindole (DAPI)	Sigma (USA)	F6057
Adhesion microscope slides	CITOGLAS (China)	80312-3161
Cadmium chloride	Sigma (USA)	655198-5G
Confocal microscope	Zeiss (Germany)	LSM700
Dust-free paper	Kimberly-Clark (USA)	34120
Inulin-FITC	Sigma (USA)	F3272
Microinjection capillaries	Zhengtianyi (China)	BJ-40	1.0 mm × 0.8 mm  × 100 mm
Micropipette beveler	NARISHIGE (JAPAN)	EG-400
OCT	SAKURA (JAPAN)	4583
Paraformaldehyde	Sigma (USA)	P6148
Pentobarbital sodium	Merck (Germany)	P11011
Shaver	Yashen (China)
Stereo microscope	Nikon (JAPAN)	SMZ1000
Sucrose	Sangon Biotech (China)	A610498
Surgical instruments	Stronger (China)		scissors, forceps, needle holder
Syringe	KDL (China)	20163150518	0.45 mm × 0.16 mm RW LB
thermostatic heater	KELL (Nanjing, China)	KEL-2010
10x TBS, pH 7.6
0.2 M Tris	Sangon Biotech (China)	A600194
1.37 M Nacl	Sangon Biotech (China)	A610476