BIOLOGY OF REPRODUCTION 64, 644–646 (2001) Testis Transplantation in Male Rainbow Trout (Oncorhynchus mykiss) James J. Nagler,1,2 Joseph G. Cloud,2 Paul A. Wheeler,3 and Gary H. Thorgaard3 Department of Biological Sciences and Center for Reproductive Biology,2 University of Idaho, Moscow, Idaho 83844-3051 School of Biological Sciences and Center for Reproductive Biology,3 Washington State University, Pullman, Washington 99164-4236 ABSTRACT The objective of the present study was to establish a procedure for the transplantation of an intact testis from one male rainbow trout (Oncorhynchus mykiss) to another individual and evaluate the reproductive function of the transplanted testis at sexual maturity. Isogenic (cloned) male rainbow trout were produced by crossing a completely homozygous male (YY) with a homozygous female (XX) to eliminate any problem of tissue rejection. Transplantation was performed on four pairs of sexually immature animals (n 5 8); each served both as a donor and recipient. The left testis was removed by making a ventral midline incision to expose the body cavity and gonads. The left testis was disconnected at the anterior and posterior points of attachment and transferred to the recipient fish where it was placed in position adjacent to the pyloric cecae. The right testis was left intact. After 4 wk, the fish were injected (i.p.) twice weekly for 8 or 9 wk with salmon pituitary extract (1.5 mg/kg) to induce precocious sexual maturation. A similar number of untreated fish were maintained as controls. Following this treatment, all the fish were killed, and the right (intact) and left (transplanted) testes were removed, weighed, and sampled for sperm. Although the mean weights of the left, transplanted testes were significantly (P , 0.05) smaller than the intact testes (transplants 5 1.2 g; intact 5 3.9 g), transplanted testes were present in all animals, had increased in mass, and were sexually mature containing sperm. The mean fertility, as measured by the proportion of eggs completing first cleavage, of sperm derived from transplanted testes (92%) was no different from the sperm obtained from intact testes (84%). Similarly, there was no difference in the number of embryos attaining the eyed stage of development, after 18 days of incubation, that were derived from transplanted (84%) or intact testes (85%). MATERIALS AND METHODS Fish A family of isogenic (cloned) male rainbow trout (Oncorhynchus mykiss) was produced by mating a homozygous female (XX) from the Oregon State University line with a homozygous male (YY) from the Swanson line [11, 12]. These fish were maintained in tanks containing dechlorinated freshwater at 14 6 18C and fed a pelleted trout ration (Rangen, Buhl, ID) at ;1.5% body weight/day. Food was withheld 1 day in advance of any handling procedures and resumed the next day. male sexual function, testes INTRODUCTION The transplantation of gonadal tissue within and between individual animals is an experimental endeavor that has a long history in reproductive biology [1–4]. Recently, the transfer of gonadal constituents between different vertebrate species has shown promise [5–7]. The ability to move gonadal tissue from one animal to another has a number of important implications relative to the conservation of reproductive potential and overcoming instances of sterility [8]. Much of the interest in performing gonadal transplantation stems from research on humans and mammalian laboratory animals. There have been no reports of the techniques required for successful gonadal transplantation in fishes. Surgical Procedure Eight fish were randomly chosen for testicular transplantation. At the time of surgery these fish were approaching 2 yr of age (;100 g in weight and 20 cm in length) but were sexually immature. Fish were anesthetized in a solution of 100 mg tricaine methanesulfonate/L water containing 100 mg sodium bicarbonate/L. Two fish were anesthetized at the same time, removed from the water, and placed in a supine position on a plastic tray. Their gills were constantly irrigated with the anesthetic solution using a pipette. The procedure began with a 4-cm midventral incision made between the pectoral and pelvic fins. The body cavities were exposed and retracted with clamps. The anterior point of attachment of the left testis (20–50 mg) in each fish was located, pulled free with forceps, and lifted from the body cavity. The posterior attachment point (i.e., sperm duct) of each testis was severed with scissors. Then each left testis was lifted free and repositioned in the same position within the recipient fish. The testes were not sutured into place but were in close contact with the body wall and parts of the Correspondence. FAX: 208 885 7905; e-mail: jamesn@uidaho.edu 1 Received: 26 June 2000. First decision: 14 August 2000. Accepted: 2 October 2000. Q 2001 by the Society for the Study of Reproduction, Inc. ISSN: 0006-3363. http://www.biolreprod.org 644 Downloaded from https://academic.oup.com/biolreprod/article/64/2/644/2723534 by guest on 19 June 2022 The major hurdle in most instances of animal transplantation is tissue rejection by the recipient animal. Inbreeding, immunosuppression, and use of tissues (e.g., brain, testis) that have a blood barrier are all strategies that have been used to overcome immune responses to allografts [4, 5, 9, 10]. In fishes, isogenic or cloned animals can be produced by either androgenesis or gynogenesis in two generations to overcome this problem [11, 12]. Isogenic fish are similar to inbred strains of mammals but have the advantage of being totally homozygous. Indeed, allografts of skin exchanged between family members were viable in isogenic rainbow trout [13]. The objective of the present study was to establish a procedure for the transplantation of an intact testis from one isogenic rainbow trout to another individual of the same family. In addition, the reproductive function of the transplanted testis was examined by obtaining sperm when sexually mature for the purposes of fertilizing eggs and following early embryonic development. The overall intent of this work is to develop an experimental research model for studies on fish reproduction. 645 TESTIS TRANSPLANTATION IN TROUT TABLE 1. Individual testis weights and fertility measures in isogenic male rainbow trout with (transplant) or without (control) left testis transplantation. Transplant fisha Testis weight (g) % Fertility % Eyed stage Control fisha Left testis Right testis Left testis Right testis 1.2 6 0.5b 92 6 5 84 6 6 3.9 6 0.6c 84 6 9 85 6 8 4.3 6 1.2 nd nd 4.6 6 1.7 nd nd a n 5 7. Mean 6 standard deviation. c Significantly different (P , 0.05) from the left testis in transplant fish; nd, not done because fish were sexually immature. b RESULTS digestive tract. The right testis was left intact in all fish. The midventral incision was closed with four or five silk (2-0 size) sutures. The surgical procedure was complete in 7–8 min, and the fish were immediately returned to their tank for recovery. Fish regained their equilibrium and commenced swimming in about 5 min. After 2 wk all fish were anesthetized as above, the healed incisions examined, and the sutures removed. Induction of Sexual Maturation Precocious sexual maturation was induced to assess whether the transplanted testis could develop to sexual maturity and produce viable sperm. A regime of injections of salmon pituitary extract (no. P3909; Sigma, St. Louis, MO) was used to induce precocial sexual maturation in all fish with a transplanted testis. Biweekly i.p. injections of 1.5 mg pituitary extract/kg fish were given beginning 4 wk after surgery. Fish were anesthetized as described above before each injection. Injections continued for 8–9 wk until testicular maturation was attained by the intact right testis. Sexual maturation was assessed by applying gentle pressure on the abdomen and observing the release of semen from the genital opening. A similar number of isogenic male fish from the same family were maintained in a separate tank as noninjected controls. Once transplant fish attained sexual maturity, all fish were killed, testes removed, and testes weighed individually. Fertility and Embryonic Development Assessments To determine whether viable sperm had been produced in the transplant fish, each testis was individually placed in a shallow plastic dish on ice and finely minced with scissors. Approximately 100 unfertilized rainbow trout eggs (Mt. Lassen Trout Farms, Red Bluff, CA) were added, mixed gently with the testicular fragments, and a small amount (20 ml) of cold trout sperm activating solution [14] was added. These dishes were immediately transferred to a coldroom at 108C to allow in vitro fertilization to proceed. After 5 min the testicular fragments were removed from the eggs, the eggs rinsed with water three times, and finally placed in a vertical fish egg incubator (Heath, Tacoma, WA). After 12 h, 10 eggs were removed, fixed in Stockards solution [15], and scored for fertilization based on the pres- Seven of the eight transplanted fish attained full sexual maturity approximately 8 wk after injections of pituitary extract began. One transplant fish died 2 wk after surgery. None of the control fish had attained sexual maturity by the time of sampling. At sampling time both the left (transplanted) and right (intact) testes in all transplant fish were swollen and white in color, indicative of sexual maturity in this species (Fig. 1). Upon dissection and removal of each testis it was apparent that the transplanted (i.e., left) testes had invariably established numerous blood vascular connections with the adjacent digestive tract, particularly the pyloric cecae. The mean weight of the right testes in the transplant fish was significantly (P , 0.05) larger, by about 2.5 times, relative to the left testes (Table 1). The mean weights of right and left testes of the control fish were similar in size and no different from the intact right testes in the transplant fish (Table 1). Both the right and left testes from the transplant fish produced sperm capable of fertilizing eggs, and there was no difference in the fertility rates between the two groups (Table 1). Similarly, there was no difference in the proportion of eyed stage embryos determined after 18 days of incubation that were derived from sperm from the left or right testes of the transplant fish (Table 1). DISCUSSION This is the first report of a successful transplantation of a testis from one fish to another. A simple surgical procedure in which an intact testis was removed from one immature rainbow trout and placed within the body cavity of another immature male fish belonging to the same isogenic family was used. The blood supply was re-established to the transplanted testis that responded to salmon pituitary extract treatment by precocially attaining full sexual maturity. Further, there was no apparent difference in the ability of sperm produced by the transplanted testes to fertilize eggs in vitro or affect embryonic development, as compared to sperm derived from the intact testis from each fish. This implies that the reproductive functionality of the transplanted testis is the same as that for the intact testis. The success of this experiment was undoubtedly due to two important features, the use of isogenic fish and their reproductive state of sexual immaturity. First, the use of a family of isogenic or cloned rainbow trout virtually elimi- Downloaded from https://academic.oup.com/biolreprod/article/64/2/644/2723534 by guest on 19 June 2022 FIG. 1. The right (upper) and left (lower) testes from a sexually mature isogenic male rainbow trout that received a transplanted testis (left) from a sibling. Bar 5 2 cm. ence of one or more cleavage divisions of the blastoderm [16]. The remaining eggs were incubated for 18 days at which time they were scored for the number showing retinal pigmentation (i.e., eyed stage). 646 NAGLER ET AL. ACKNOWLEDGMENT The authors thank Mr. Jerry Bouma for his assistance with the fish husbandry. REFERENCES 1. Grigorieff W. Die schwangerschaft bei der transplantation der eierstocke. Zbl Gynaekol 1897; 21:663–668. 2. Parkes AS. Survival time of ovarian homografts in two strains of rats. J Endocrinol 1956; 13:201–210. 3. Lara HE, Dees WL, Hiney JK, Dissen GA, Rivier C, Ojeda SR. Functional recovery of the developing rat ovary after transplantation: contribution of the extrinsic innervation. Endocrinology 1991; 129:1849– 1860. 4. Barten EJ, Garybian H, Klopper PJ, Newling DWW. Homologous testis transplantation in dogs. Transplant Int 1997; 10:362–368. 5. Russell LD, Brinster RL. Ultrastructural observations of spermatogenesis following transplantation of rat testis cells into mouse seminiferous tubules. J Androl 1996; 17:615–627. 6. Ogawa T, Dobrinski I, Avarbock MR, Brinster RL. Xenogeneic spermatogenesis following transplantation of hamster germ cells to mouse testes. Biol Reprod 1999; 60:515–521. 7. Dobrinski I, Avarbock MR, Brinster RL. Transplantation of germ cells from rabbits and dogs into mouse testes. Biol Reprod 1999; 61:1331– 1339. 8. Ogawa T, Arechaga JM, Avarbock MR, Brinster RL. Transplantation of testis germinal cells into mouse seminiferous tubules. Int J Dev Biol 1997; 41:111–122. 9. Borlongan CV, Cameron DF, Saporta S, Sanberg PR. Intracerebral transplantation of testis-derived Sertoli cells promotes functional recovery in female rats with 6-hydroxydopamine-induced hemiparkinsonism. Exp Neurol 1997; 148:388–392. 10. Coddington DA, Lawen JG, Yang H, O’Hali W, Wright JR Jr. Xenotransplantation of fish islets into the non-cryptorchid testis. Transplant Proc 1997; 29:2083–2085. 11. Young WP, Wheeler PA, Fields RD, Thorgaard GH. DNA fingerprinting confirms isogenicity of androgenetically-derived rainbow trout lines. J Hered 1996; 87:77–81. 12. Robison BD, Wheeler PA, Thorgaard GH. Variation in development rate among clonal lines of rainbow trout (Oncorhynchus mykiss). Aquaculture 1999; 173:131–141. 13. Ristow SS, de Avila JM, Baldwin TJ, Wheeler PA, Thorgaard GH. Acceptance of skin grafts by isogenic trout (Oncorhynchus mykiss). Am J Vet Res 1996; 57:1576–1579. 14. Nilsson EE, Cloud JG. Extent of mosaicism in experimentally produced diploid/triploid chimeric trout. J Exp Zool 1993; 266:47–50. 15. Gray AK, Evans MA, Thorgaard GH. Viability and development of diploid and triploid salmonid hybrids. Aquaculture 1993; 112:125– 142. 16. Ballard WW. Normal embryonic stages for salmonid fishes, based on Salmo gairdneri Richardson and Salvelinus fontinalis (Mitchell). J Exp Zool 1973; 184:7–26. 17. Kennedy-Stoskopf S. Immunology. In: Stoskopf MK (ed.), Fish Medicine. Philadelphia: WB Saunders; 1993: 149–159. Downloaded from https://academic.oup.com/biolreprod/article/64/2/644/2723534 by guest on 19 June 2022 nated any immune reaction against the transplanted testis. Tissue rejection is well known in a wide range of fishes due to the presence of a major histocompatibility complex [17]. Secondly, these fish were sexually immature with relatively small testes at the time of surgery. The overall mass of the transplanted testis was small (e.g., 20–50 mg) with a large surface area to mass ratio. This is presumably advantageous for the rapid development of a blood supply with adjacent tissues, like the digestive tract, before significant tissue necrosis occurred. The smaller size of the transplanted testis at the time of sampling is probably a result of the time interval necessary for development of a new blood supply, during which the right, intact testis continued to grow. One acknowledged drawback of this procedure is that access to the transplanted testis requires reopening the body cavity. However, the simplicity and postoperative survival (seven of eight fish) of the present surgical procedure vastly outweighs the difficulty of the microsurgery needed to reconnect tiny sperm ducts in fish of this small size. The testis transplantation procedure in the present study using isogenic rainbow trout will be a useful research model in fishes. A number of important applications derive from this research tool. It will be invaluable in experimental studies to determine the mechanism of action of environmental contaminants during reproductive development, by permitting the hypothalamic-pituitary axis to be separated from the gonads. This research model will also be used for studies on the cryopreservation of fish testes or ovaries and their viability upon return to a recipient fish. The possibility of storing frozen gonadal fragments for later transferal to donor animals is a potentially important strategy for the preservation of the genetics of endangered fish populations.