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 epithelium into a basal and an adluminal compartment. During stages VIII - IX of the epithelial cycle, preleptotene/leptotene spermatocytes from the basal compartments migrate across the BTB, entering the adluminal compartments³. Therefore, the function of the BTB is to provide an immunoprivileged microenvironment for the completion of meiosis and spermiogenesis^4,5,6. Unlike other blood-tissue barriers (e.g., blood-brain barrier) that are only composed of tight junctions (TJs), the BTB is formed by four different junctions (TJs, ectoplasmic specializations, gap junctions, and intermediate filament-based desmosomes) between Sertoli cells^1,7.

Many studies have used genetically-modified mice, virus infections, and environmental toxicants to investigate mechanisms of BTB integrity^7,8,9. The BTB disruption induces impaired spermatogenesis and subfertility or infertility. Since the BTB formation and integrity have been confirmed to be affected by contacts between Sertoli cells⁸, an in vitro model based on primary culture of isolated Sertoli cells has been used for BTB study. However, this model cannot accurately mimic BTB dynamics in vivo. Moreover, no such co-culture of germ cells with Sertoli cells has been established as capable of reflecting all relevant structural and functional components of the BTB^10,11.

In general, in vivo BTB integrity assays are typically based on small molecules, such as EZ-Link Sulfo-NHS-LC-Biotin and FITC-conjugated inulin (inulin-FITC). Normally, the diffusion of biotin or inulin-FITC from the basal compartment is blocked by BTB structure. Therefore, we are able to use this method to assess the extent of BTB damage compared with control groups. While BTB can be compromised with certain types of stimuli, such as treatment with cadmium chloride (CdCl₂)¹², BTB becomes accessible to small molecules, which eventually enter the adluminal compartment as indicators.

An early in vivo BTB integrity assay involves injecting biotin or inulin-FITC into the jugular vein, which involves surgery, and is invasive, complicated, and time-consuming. Besides, as the reporter substances diffuse through the whole body via the circulation, the local concentration of biotin or inulin-FITC in the seminiferous tubules is limited. Moreover, systemic exposure may induce immune reactions. Here, we present a simple and effective in vivo BTB integrity assay enabling direct injection of a small aliquot of inulin-FITC into the interstitium of a testis. Using the fluorescent labeling method, the staining process is convenient, as secondary antibodies are not required. Here, the process of fluorescent dye entering the testis is visualized.

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 forceps to use to obtain capillaries of a 50-µm diameter at the tip.
      Note: The tips may be too long to penetrate the testis.
   4. Sharpen the tip in a 30° angle by using a micropipette beveler (Figure 1C).
2. Reagents
   1. Prepare 1% pentobarbital sodium: dissolve pentobarbital sodium 100 mg in 10 mL of phosphate-buffered saline (PBS) (step 2.3.3).
   2. Prepare 10 mg/mL inulin-FITC: dissolve inulin-FITC 1 mg in 100 μL of PBS (step 2.1.6).
   3. Prepare 4% paraformaldehyde: dissolve paraformaldehyde (PFA) 4 g in 100 mL of PBS (step 2.3.3).
   4. Prepare 30% sucrose: dissolve sucrose 3 g in 10 mL of PBS (step 2.3.4).
   5. Prepare 0.1% cadmium chloride: dissolve cadmium chloride 10 mg in 10 mL of PBS (step 2.1.1).

2. Methods

1. Anesthesia and presurgery preparation
   1. Weigh 8-week-old C57BL/6 male mice and calculate the required dose of CdCl₂. Treat a group of mice (n = 3) with CdCl₂ (5 mg/kg b.w., i.p.) for 3 d before surgery. Treat another group of 8-week-old C57BL/6 male mice (n = 3) with PBS as control.
   2. Perform surgery under aseptic conditions by using sterile syringe, needle, scissors, and forceps.
   3. Prepare anesthesia working solution freshly. The required dose of anesthesia is 70 mg of pentobarbital sodium/kg of body weight. On average, an adult C57BL/6 mouse at 8 weeks of age weighs ~25 g, which corresponds to 175 μL of 1% pentobarbital sodium (1.75 mg per mouse).
      NOTE: The pentobarbital sodium is dissolved in sterile phosphate-buffered saline (PBS). The solution is filtered by 0.22 um filter unit before being used.
   4. Clean the area for surgery with 75% ethanol and cover the area with a clean tissue towel.
   5. Turn on the thermostatic heater and adjust the temperature to 37 °C.
   6. Prepare inulin-FITC working solution (10 mg/mL) on the day of the surgery.
2. Surgical procedure
   1. Weigh 8-week-old C57BL/6 male mice and calculate the required dose of anesthesia.
   2. Perform an intraperitoneal injection of pentobarbital sodium using the 1-mL sterile syringe. Keep the mouse in a clean cage.
   3. Observe the eye reflex response and breathing pattern of the mouse to confirm that it is under complete anesthesia.
      NOTE: Usually, 10 - 15 min are needed until the mouse is deeply anesthetized, with a total lack of toe pinch response and maintenance of slow, steady breathing.
   4. Move the mouse to the operation area and cover its eye area with a moist tissue paper to avoid dryness during anesthesia (Figure 2A).
   5. Shave its abdominal hair with a shaver and disinfect the surgery area with 75% ethanol.
   6. With sharp scissors, make a 1-cm skin incision above the preputial glands to expose the abdominal wall. Lift the abdominal wall with small forceps and make a 0.5-cm incision to expose the peritoneal cavity (Figure 2B).
   7. Use forceps to search for fat pads around the epididymis and testis. Carefully pull the fat pads out to expose the attached testis clearly (Figure 2C and 2D). Usually, operate on one testis at a time.
      NOTE: Avoid touching the fat pads and testis with hands.
   8. Place a paper (9 cm in diameter) underneath the fat pads and testis (Figure 2C).
   9. Inject inulin-FITC into a microinjection pipette and connect it to the micromanipulator unit (Figure 1D). Gently insert the microinjection pipette under the tunica albuginea and load a total of 20 μL of inulin-FITC into the interstitium of the testis (Figure 2E and 2F).
      NOTE: Carefully depress the microinjection pipette to avoid moving it.
   10. Monitor the movement of inulin-FITC in the testis (Figure 2G).
   11. Immediately put the testis back into the abdominal cavity after the completion of the injection.
   12. Repeat the procedure on the contralateral testis. This testis is injected with 20 μL of PBS as a control.
   13. Close the skin with a surgical suture and move the mouse to a heat pad of a thermostatic heater (Figure 1B). Administer a dose of 1 mg of analgesic (buprenorphine) per 1 kg of body weight via subcutaneous injection.
3. Harvesting the testes and frozen sections preparation
   1. 40 min after the injection, euthanize the recipient mouse by cervical dislocation according to animal care and use guidelines.
   2. Use a pair of sharp scissors to collect the testes. Place the testes into 1 mL of ice-cold PBS to remove any blood contamination.
   3. Fix the testes in 4% paraformaldehyde (PFA) at 4 °C for 12 - 24 h. Discard the 4% PFA and wash the tissue 3x with 1.5 mL of 1% PBS at room temperature.
   4. Dehydrate the tissue in 30% sucrose overnight. Put the testis in the embedding frame and cover the tissue with optimal cutting temperature compound (OCT). After the OCT is frozen, fill the embedding frame with OCT so that the whole testis can be covered with OCT.
   5. Cut 5-μm-thick, frozen cross sections of the testes in a cryostat at -20 °C and let them adhere to microscope slides.
      NOTE: Refreezing embedded tissues in dry ice may increase the rigidity for cutting.
4. Image requisition
   1. Place the slides in a humidified box. Warm the slides at room temperature for 10 min.
   2. Wash the sections 3x with Tris-buffered saline (TBS) at room temperature.
   3. Air-dry for 5 min in the dark. Wipe off any residual TBS with dust-free paper.
   4. Cover the cross sections with 4’,6-diamidino-2-phenylindole (DAPI). Place the inverted coverslip on a microscope slide.
   5. Acquire images using a confocal microscope. A total of 20X magnification is generally sufficient for detecting the brightly fluorescent signal.

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

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