Development of heterotopic transplantation of the testis with the epididymis to evaluate an aspect of testicular immunology in rats

Abstract

Transplantation of testicular cells and tissues has been studied for the investigation of immunology of the testis, which is an immunologically privileged organ. However, reports of transplant of the testis at organ level have been extremely limited because of technical difficulties of the orthotopic testis transplantation (OTT) in experimental animals. In the present study, we developed a new and simple model of the heterotopic testis transplantation (HTT), which is donor testis transplantation into the cervical region of recipients, in a syngeneic model in rats [donor Lewis (LEW) graft to LEW recipient]. The duration of HTT was significantly shorter and success rate higher than that of OTT. To histologically evaluate HTT, the local immune responses were compared among the syngeneic model, an acute rejection allogeneic model [donor Augustus Copenhagen Irish (ACI) graft to LEW recipient] and a chronic rejection allogeneic model (donor F344 graft to LEW recipient) at postoperative day 3. We found that allogeneic ACI grafts resulted in mild and not severe orchitic lesions, whereas immune responses of allogeneic F344 grafts seemed intact and were not significantly different from those of syngeneic LEW grafts. These results suggest that our new operative procedure will be useful in future for the investigation of the testicular immunology.

Fig 1. Surgical procedures for cervical heterotopic testis transplantation (HTT).

A) The exposed testis with the epididymis and a part of the vas deferens for harvest in a donor rat. B) The harvested organs with nutrient vessels. C) Two cuffs were attached to the recipient’s common carotid artery and external jugular vein. D) The desquamated recipient’s skin for folding the transplanted testis. E) The reperfusion after cervical HTT in a recipient rat. F) The placement of the testis into the subcutaneous dorsal region of the neck in a recipient rat.

Fig 2. Schematic overview of cervical heterotopic testis transplantation (HTT) and subcutaneous temperature.

A) The technique for harvesting the testis with the epididymis and a part of the vas deferens from a donor rat. B) The technique for cervical HTT with the cuff technique in a recipient rat. C) The fitting of the testis into the subcutaneous dorsal region of the neck and draining of the vas deferens outside the body in a recipient rat. D) The subcutaneous temperatures of rats were measured in the scrotal (32.1 ± 1.4°C, n = 6), cervical (31.9 ± 1.2°C, n = 6), and abdominal cavities (36.7 ± 0.2°C, n = 6). AA: abdominal aorta, B: bladder, CCA: common carotid artery, EJV: external jugular vein, K: kidney, VD: vas deferens, IVC: inferior vena cava, T: testis, TA: testicular artery, TV: testicular vein; n.s.: not significant, *P < 0.05.

Fig 3. Histological feature of the arterioles inside the testis.

A, sham group. B, heterotopic testis transplantation (HTT) group. C, orthotopic testis transplantation (OTT) group. D, vasectomy (Vx) group. E, ischemia (IS) group. F, cryptorchidism (CR) group. The arterioles of the HTT, OTT, Vx, and CR groups exhibited a normal appearance, including numerous red blood cells, that was similar to the sham group. In the IS group, only eosinophilic materials were observed inside the arteriole, and the vascular wall displayed degeneration. Black bar: 100 μm.

Fig 4. Histological sections of the testes.

A, sham group (n = 6). B, heterotopic testis transplantation (HTT) group (n = 6). C, orthotopic testis transplantation (OTT) group (n = 6). D, vasectomy (Vx) group (n = 6). E, ischemia (IS) group (n = 6). F, cryptorchidism (CR) group (n = 6). G, Johnsen’s score for spermatogenesis. *P < 0.05 vs. HTT group. **P < 0.01 vs. HTT group. Black bar: 100 μm.

Fig 5. Histological sections of the epididymis.

A, sham group. B, heterotopic testis transplantation (HTT) group. C, orthotopic testis transplantation (OTT) group. D, vasectomy (Vx) group. E, ischemia (IS) group. F, cryptorchidism (CR) group. The HTT and OTT groups had normal epididymal ducts and intra-duct compartments. The Vx group showed many retained spermatozoa in the epididymal ducts. The IS group showed coagulation necrosis of the epididymal ducts and adjacent interstitium. The CR group showed many deformed germ cells and absence of spermatozoa in the epididymal ducts. Black bar: 100 μm.

Fig 6. Ki67 immunohistochemistry of the testes.

A, sham group (n = 6). B, heterotopic testis transplantation (HTT) group (n = 6). C, orthotopic testis transplantation (OTT) group (n = 6). D, vasectomy (Vx) group (n = 6). E, ischemia (IS) group (n = 6). F, cryptorchidism (CR) group (n = 6). G, the number of seminiferous tubules with Ki67-positive cells per 100 round and oval-shaped seminiferous tubules was determined in each testis. In all groups, Ki67-positive germ cells were observed along the basement membrane of the seminiferous tubules. STs: seminiferous tubules. *P < 0.05 vs. HTT group. **P < 0.01 vs. HTT group. Black bar: 100 μm.

Fig 7. Histological and Ki67 immunohistochemical sections of the grafted testes.

The LEW (A, D), ACI (B, E), and F344 (C, F) groups (n = 6). Johnsen’s score for spermatogenesis (G). The number of seminiferous tubules with Ki67-positive cells per 100 round and oval-shaped seminiferous tubules was determined in each testis (H). The score and number of Ki67-positive cells in the ACI group significantly decreased compared with that in the LEW group. STs: seminiferous tubules. *P < 0.05 vs. LEW group. **P < 0.01 vs. LEW group. Black bar: 100 μm.

Fig 8. Histological and immunohistochemical sections of arterioles of the grafted testes.

CD4 and CD8 immunohistochemistry in the grafts of the LEW (A, D, G), ACI (B, E, H), and F344 (C, F, I) groups. Histopathological changes were evaluated by counting the number of infiltrating CD4⁺ and CD8⁺ cells per mm² of the testicular interstitium (J, K). Injuries to the vascular endothelium and significant infiltration of CD4⁺ and CD8⁺ lymphocytes around the arterioles were found in the ACI group. The arrow heads indicate lymphocytes. *P < 0.01 vs. LEW groups. Black bar: 50 μm.

Fig 9. mRNA levels in the grafted testes detected by real time RT-PCR.

In the ACI group, mRNA expression levels of inflammatory mediators, such as IFN-γ, IL-1β, and IL-10, and apoptosis-related genes, such as caspase 3 and caspase 8, were significantly higher than in the LEW group (n = 3 for each group). *P < 0.05 vs. LEW group. **P < 0.01 vs. LEW group.