British Jorrrml of Urology (1973) 45, 696-701

Transplantation of Interstitial Cells of the Testis MILES FOX, PAULINE F. BOYLE and JOHN C. HAMMONDS

Department of Urology, Royal Hospital, Shefield

Attempts to transplant the testis have been made since John Hunter’s time, but numerous factors have prevented success. These included ignorance of aseptic techniques initially, difficulties with vascular anastomoses when whole organ transplantation was employed (Attaran and Hodges, 1966; Puchetti, 1966) and problems with immunological rejection mechanisms. However, work carried out using individual components of the testis has produced more encouraging results. Turner (1938) reported transplantation of seminiferous tubules and interstitial cells of the testis in intra-ocular sites in the rat and Williams (1950) transplanted similar tissues in millipore- type chambers in the ear of the rabbit, and showed some proliferation of the component cells.

The present experiments were devised to study the fate of free testicular grafts and their effects on primary and secondary sexual development. Homogenate suspensions of the testis containing liberated interstitial cells were grafted into various sites in castrated animals. The effects were observed in autologous and allogeneic combinations in inbred strain mice, and the survival of the cells was assessed histologically.

Materials and Methods

Transplantation was performed between (CBA-T6T6 x C57BL) F1 hybrid mice, and between the F1 hybrids as donors and C57BL strain animals as recipients, at the ages of 3 weeks in one group of experiments and 3 months in another group.

Bilateral orchidectomy was carried out under ether anaesthesia through a midline vertical lower abdominal transperitoneal incision. The testes were placed in a sterile petri dish with 3-4 drops of normal saline and cut up finely with scissors. A thick suspension was made of the con- tents of both testes and aspirated into a brain-type cannula by slowly withdrawing the plunger. The homogenate was subsequently injected into localised areas in ear pinnae and under the skin of the back and snout. The animals were weighed weekly over a period of 3 months and groups were killed at varying intervals to determine the weight, appearance and histological structure of the genital apparatus, particularly the seminal vesicles and penis. The graft areas were examined for vascularisation, sectioned, and stained with haematoxylin and eosin for histological study. Observations were also made in groups of castrated non-transplanted animals, and sham operated control mice in which the same operation was performed as above, apart from ligating the sper- matic cords and removing the testes.

Results

Castrated animals gained weight more slowly than sham operated ones over the 3-month period studied (Fig. l), the differences becoming apparent from the 1st week and statistically significant from the second. Following autologous grafting of the testes into the subcutaneous dorsal site, weight gains corresponded to those of sham operated mice from the 3rd week (Fig. 1). Read at the 29th Annual Meeting of the British Association of Urological Surgeons in London, June 1973.

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Fig. 3. Relative seminal vesicle weights of adult (C57BL x CBA-T6T6) F, hybrid mice, 3 months after operation.

698 BKITISH JOURNAL OF UROLOGY

Seminal vcsicle weights are expressed as an arbitrary convenient ratio of the weight of thc vesicles to the body weight of the animal:

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Following bilateral orchidectomy in adolescent animals, the seminal vesicles did not develop and in adults they atrophied. When testicular suspensions were implanted into the animals, development was restored, but to varying degrees according to the site employed (Figs. 2 and 3). The subcutaneous dorsal site was the most effective, relative seminal vesicle weights approximating to those of controls 3 months after grafting. Subcutaneous injection into the snout produced slightly less enlargement of the vesicles and injection into the ear pinnae least of all. Howevcr, even in the latter sitc, thcre was a statistically significant increase over the vesicle weights of

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castratcd mice with only a fcw individual rcsults overlapping. Seminal vesicles from castrated animals contained little or no secretion, whereas thosc from normal, sham operated and trans- planted animals showed distension of the collecting spaces with secretory material. Changes in the size and weight of the penis followed thosc of the seminal vesicles (Fig. 4).

The graft areas, 3 months postoperativcly, were seen as light pink localised swellings which had obtained a good blood supply from surrounding structures. The blood vessels were easily visible with the naked eye and were prominent in many cases (Fig. 5). On histological examination there w;is initially some round cell infiltration with proliferation of fibroblasts and occasional giant cell formation. Seminiferous tubules were atrophic in all cases and there was no evidence of spermatogenesis. However, interspersed between this tissue were small groups of larger cells containing granular type cytoplasm which stained pinkcr with haematoxylin and eosin than the surrounding structures (Fig. 6) . These cells were indistinguishable from those of normal mouse interstitial cells. Mitoses were not seen but the impression was obtained that the clumps becamc larger with progression of time over the 3-month period studied.

TRANSPLANTATION OF INTERSTITIAL CELLS OF THE TESTIS

Fig. 5. Free testicular graft, subcutaneous dorsal site 3 months after transplantation, showing a good blood supply from surrounding tissues.

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Fig. 6. Group of interstitial cells from a subcutaneous graft 3 months after transplantation.

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In a preliminary group of experiments observations made in adult mice 7 weeks after castration followed by allotransplantation showed atrophy of seminal vesicles similar to those of control castrated animals, while in autotransplanted mice at the same period the seminal vesicles were considerably larger (Fig. 7). A yellowish swelling was present in the area of the allograft with no obvious vascularisation from the surrounding tissues and no cells resembling interstitial cells were found on histological examination.

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Discussion

The results of the experiments have shown that following castration of young mice, total body weights increase more slowly than those of normal animals and that development of the seminal vesicles and the penis is markedly impaired. Restoration to normal values was obtained after subcutaneous autotransplantation of testicular homogenates, the grafts became vascularised and cells indistinguishable from normal interstitial cells were a constant feature on histological examination. Even though direct evidence of endocrine secretion of either androgens or gonado- trophins were not available in these experiments, no alternative explanation to the observed facts is likely other than that actively secreting interstitial cells were transplanted. Transplantation under the skin of the back of the animals produced better development of seminal vesicles than in the possibly more restricted sites under the skin of the snout or in the ear pinnae. Although cells resembling interstitial cells were seen in all the sites studied, it is likely that the cells were freer to divide and possibly received a better blood supply in the looser subcutaneous region of the back.

Spermatogenesis was not seen in any of the sections examined. Seminiferous tubules rapidly atrophied, their component cells died and were phagocytosed. This pattern does not occur when

TRANSPLANTATION OF INTERSTITIAL CELLS OF THE TESTIS 70 1

transplantation of the whole organ with vascular anastomoses is performed (Lee, Tung and Orloff, 1971). Sperm forming cells are probably not able to obtain sufficient oxygen or nourishment for survival from interstitial fluid when transplanted in the “free” state before a blood supply is formed, and consequently atrophy.

The whole graft was found to be atrophic in animals sacrificed 7 weeks after allotransplantation, and seminal vesicle weights were the same as those of purely castrated mice. Immunological re- jection is likely to have been responsible, since immunosuppressive therapy was not employed in this group of experiments. Whether or not rejection of interstitial cells is delayed in the presence of endocrine “need” is being investigated, but from these experiments it is evident that at 7 weeks after grafting there was no evidence of them or of their activity.

Summary

Free transplantation of testis homogenates was performed in the mouse in autologous combi- nations.

Results were assessed histologically and by the effect on primary and secondary sexual charac- teristics.

While seminiferous tubules atrophied and no spermatogenesis occurred, interstitial cells sur- vived.

In previously castrated animals transplantation was followed by normal seminal vesicle and penis development in adolescent mice and prevention of atrophy in adult animals.

These changes were not seen after allotransplantation, presumably due to graft rejection.

We are grateful to the Medical Research Council for supporting this work, to Dr Laurence Henry for assistance with histological studies and the photographic department of the Royal Hospital, Sheffield for the illustrations.

References

ATTARAN, S. E. and HODGES, C. V. (1966). Technique for testicular transplants. Investigative Urology, 4, 390-394. LEE, S., TUNG, K. S. K. and ORLOFF, M. J. (1971). Testicular transplantation in the rat. Transplantation Proceedings,

PUCHETTI, V. (1966). Trapianti Autoed Omoplastici De Testicolo Corn Immediata Connessioni Vascolare. Studio

TURNER, C. D. (1938). Intra-ocular homotransplantation of prepuberal testes in the rat. American Journal of

WILLIAMS, R. G. (1950). Studies of living interstitial cells and pieces of seminiferous tubules in autogenous grafts

3, 586-590.

Sperimentale. Chirurgiu Ztuliana, 18, 123-1 38.

Anatomy, 63, 101-159.

of testis. American Journal of Anatomy, 86, 343-369.

The Authors

Miles Fox, MD, ChM, FRCS, Consultant Urological Surgeon. Pauline, F. Boyle, BSc, Scientific Officer. John C. Hammonds, FRCS, Registrar in Urology.