Protocols for germ cell transplantation and testis tissue xenografting are described. Germ cell transplantation results in donor-derived spermatogenesis in recipient testes and represents a functional reconstitution assay for identification of spermatogonial stem cells (SSCs). Testis tissue xenografting reproduces testis development and spermatogenesis of various donor species in recipient mice.
Germ cell transplantation was developed by Dr. Ralph Brinster and colleagues at the University of Pennsylvania in 19941,2. These ground-breaking studies showed that microinjection of germ cells from fertile donor mice into the seminiferous tubules of infertile recipient mice results in donor-derived spermatogenesis and sperm production by the recipient animal2. The use of donor males carrying the bacterial β-galactosidase gene allowed identification of donor-derived spermatogenesis and transmission of the donor haplotype to the offspring by recipient animals1. Surprisingly, after transplantation into the lumen of the seminiferous tubules, transplanted germ cells were able to move from the luminal compartment to the basement membrane where spermatogonia are located3. It is generally accepted that only SSCs are able to colonize the niche and re-establish spermatogenesis in the recipient testis. Therefore, germ cell transplantation provides a functional approach to study the stem cell niche in the testis and to characterize putative spermatogonial stem cells. To date, germ cell transplantation is used to elucidate basic stem cell biology, to produce transgenic animals through genetic manipulation of germ cells prior to transplantation4,5, to study Sertoli cell-germ cell interaction6,7, SSC homing and colonization3,8, as well as SSC self-renewal and differentiation9,10.
Germ cell transplantation is also feasible in large species11. In these, the main applications are preservation of fertility, dissemination of elite genetics in animal populations, and generation of transgenic animals as the study of spermatogenesis and SSC biology with this technique is logistically more difficult and expensive than in rodents. Transplantation of germ cells from large species into the seminiferous tubules of mice results in colonization of donor cells and spermatogonial expansion, but not in their full differentiation presumably due to incompatibility of the recipient somatic cell compartment with the germ cells from phylogenetically distant species12. An alternative approach is transplantation of germ cells from large species together with their surrounding somatic compartment. We first reported in 2002, that small fragments of testis tissue from immature males transplanted under the dorsal skin of immunodeficient mice are able to survive and undergo full development with the production of fertilization competent sperm13. Since then testis tissue xenografting has been shown to be successful in many species and emerged as a valuable alternative to study testis development and spermatogenesis of large animals in mice14.
PART A. Germ cell transplantation in mice
1. Preparation of recipient mice
2. Preparation of microinjection pipettes
3. Preparation of donor cells for transplantation
4. Transplantation procedure (Figure 1)
5. Analysis of the recipient testes
PART B. Ectopic xenografting in mice
(From Dobrinski and Rathi 200815, and Rodriguez-Sosa et al. 201116).
1. Collection of donor tissue
2. Preparation of donor tissue
3. Castration of recipient mouse
4. Ectopic xenografting
5. Collection of testis xenografts for analysis and sperm harvesting
PART C. Representative Results
1. Germ cell transplantation
If the transplanted donor cell suspension contains spermatogonial stem cells carrying a genetic marker such as the LacZ transgene, colonization of donor SSCs in the recipient testis can be visualized by X-gal staining as distinctive blue segments of the seminiferous tubules after 2-3 month post-transplantation3 (Figure 2A). A well- established colony should have a long dark blue stretch of completely filled segments with two or more layers of blue cells closer to the center, and relatively weaker stained regions at both ends where a network of single, paired or small groups of blue cells is apparent3 (Figure 2B). A cross section of the dark blue seminiferous tubule region should reveal well-established and well-organized spermatogenesis with blue germ cells at various differentiation stages (Figure 2C).
2. Testis tissue xenografting
The viability of testis tissue after transplantation is inversely correlated with the developmental stage of the donor; the best outcome is obtained when tissue from newborn males is used, while adult tissue shows a high tendency to degenerate and die17-21. Generally, xenograft success decreases as donor tissue undergoes meiosis of the first wave of spermatogenesis. Time to full development of immature donor tissue and complete spermatogenesis is species specific, and often somewhat shorter in comparison with testes in situ. The number of seminiferous tubules with complete spermatogenesis is variable according to the species. While in sheep, goats and pigs that number is greater than 50%, in cattle and cats it is less than 15% 14(Figure 3).
Figure 1. Germ cell transplantation procedure. Place recipients in dorsal recumbency after anaesthesia and make a ~0.4-inch midline abdominal incision (A). Expose testis by withdrawing the fat pad attached to the epididymis and testis and place a thin sterile drape underneath the fat pad/testis for better visual identification (B). Position the testis and epididymis so that the efferent ducts buried in the fat pad are discernible (C, D, E). Identity the efferent ducts and gently remove fat tissue around the ducts. A piece of colored paper or plastic can be placed underneath the ducts for better visualization (G). Break or grind the pipette tip according to the size of the ducts and load cell suspension into the pipette (H). Carefully insert the pipette into a duct in the bundle of efferent ducts, gently thread a few mm toward the testis (the arrow in H shows the direction of pipette injection and threading). A testis with successful injection into the seminiferous tubules is shown (I). Ts: Testis; Ep: epididymis.
Figure 2. Representative results for germ cell transplantation A) An injected testis stained with X-gal. The blue segments of the seminiferous tubules represent established colonies from transgenic donor SSCs (LacZ transgene). B) Higher magnification of a SSC colony at 3 month post-transplantation. The dark blue region in the center represents complete spermatogenesis and the pale blue regions at the ends represent growing extension of the colony. C) A histological section of the dark blue seminiferous tubule (3 month post-transplantation) shows well organized spermatogenesis. Scale bars in B and C are 200 μm and 30 μm, respectively. (Figures adapted from Annu Rev Cell Dev Biol. 2008;24:263-86 and Biol Reprod. 1999 Jun;60(6):1429-36).
Figure 3. Ectopic xenografting of immature testis tissue from large animals into immunodeficient mice. Fragments of immature donor testis (~1 mm3) transplanted under the dorsal skin of immunodeficient mice (A) are able to survive and respond to mouse gonadotropins. As a result, testis tissue undergoes complete development, including formation of fertilization competent sperm (B). Once testis xenografts are collected (C) they can be used for analysis or to obtain sperm (D) for ICSI (E) and embryo production (F). Bars equal 50 μm (B, E and F) or 10 μm (D). (Modified from Rodriguez-Sosa and Dobrinksi 200914).
1. Germ cell transplantation
Germ cell transplantation provides the only functional assay for unequivocal confirmation of the presence of spermatogonial stem cells (SSCs) in a cell population. Only SSCs can home to and colonize the SSC niche at the basement membrane and initiate donor-derived spermatogenesis. Germ cell transplantation made it possible to study and manipulate SSCs in an unprecedented manner. The technique has been used to produce transgenic animals through genetic manipulation of SSCs4; to elucidate the pattern, efficiency and kinetics of SSC colonization3,8; to study the signalling pathways that regulate SSC self-renewal and differentiation9,10; to characterize surface markers on SSCs for their identification22,23; to study the niche environment for SSCs6,7,24. Furthermore, reciprocal transplantation has been employed to investigate whether a phenotype of infertility originated from a defect in Sertoli cells or in germ cells25,26.
For transplantation to work efficiently, choice and treatment of recipient animals is important. Recipients should be either genetically matched to donors or immune-suppressed. Recipients should also lack endogenous spermatogenesis: either due to a mutation as in W/ Wv mice, or rendered infertile as a result of germ cell depletion by irradiation or chemotherapeutic drugs such as Busulfan. Moreover, a good preparation of donor cell suspensions and proficiency in transplantation procedure are important for the success of the technique as well.
Germ cell transplantation has its own limitations. There is no fast read-out for results. The analysis of recipient testes needs to wait at least two month as reestablishment of complete spermatogenesis in the otherwise infertile recipients happens two month after transplantation3. It is a qualitative or semi-quantitative assay due to large variations in cell number injected and degree of recipient germ cell suppression. Although the concept of germ cell transplantation has been adapted to other animal species, the procedure itself is technically different and somewhat more challenging in non-rodent species as a result of the anatomic differences across species11.
2. Testis tissue xenografting
Testis tissue xenografting works across many mammalian donor species and is a relatively simple technique. As in other types of transplantations, the sooner after collection the tissue is transplanted the bigger chance of success. Therefore, preservation and handling of the tissue from collection to transplantation is important. However, in our experience testis tissue does not require special handling other than being kept refrigerated. Testis tissue can be maintained at refrigerator temperature up to 24 – 36 hr, and then fragments can be prepared for transplantation. Furthermore, fragments of fresh testis can be maintained in standard culture medium at 4 °C overnight prior transplantation without a noticeable effect on the grafting outcome27. Testis tissue can also be cryopreserved if long-term storage is desired. Studies performed in goat13, pig13,27, and monkey28 have shown that freezing and subsequent thawing of testis tissue does not affect significantly its capability to develop and produce sperm after ectopic xenografting in mice. Successful cryopreservation of testis tissue can be achieved by automated-freezing28 or conventional slow-freezing in an alcohol bath13,27, using DMSO as cryoprotectant in standard tissue culture medium containing FBS. For transplantation, cryopreserved testis tissue is then thawed by standard methods and subsequently washed in culture medium before transplantation27. Once the tissue has been transplanted the recipient mouse serves as an in vivo incubator and no major interventions are required. However, in some cases supplementation with exogenous gonadotropins may be required; testis tissue from 6-month-old rhesus monkeys required injecting recipient mice subcutaneously with 10 IU of hCG twice a week to attain full spermatogenesis at 6-7 months29.
As mentioned above, the best outcome is obtained when tissue from newborn males is used. Tissue from males in which postmeiotic germ cells are present shows a tendency to degenerate. However, with immature animals complete recapitulation of testis development is possible and has numerous clinical and research applications. In a clinical setting, testis tissue xenografting can be used for fertility preservation, particularly in immature males in which sperm recovery is not an option. Small pieces in the form of biopsies can be collected and frozen for long-term storage. When desired, the fragments can be thawed and grafted into mice27,28. Another alternative is the cryopreservation of the sperm that is harvested from xenografts. Microinjection with snap-frozen sperm from pig testis xenografts resulted in generation of morphologically normal embryos, although at a lower efficiency in comparison to testicular, epididymal, or ejaculated sperm27. After tissue has developed, it can be collected for harvesting sperm and sperm can be used to produce embryos in vitro13,16,30. A limitation of this, however, is the fact that resulting sperm do not undergo epididymal maturation and therefore require ICSI for fertilization. Therefore, use of xenograft-derived sperm for fertilization is limited to species where ICSI has been established.
In research, testis tissue xenografting is an attractive alternative to study testis development and spermatogenesis of large species in a rodent model. For example, a single donor testis can be transplanted to multiple mice. Recipient mice can then be exposed to different treatments, and/or sacrificed at different time points for xenograft collection. This not only eliminates donor effects, but also reduces the number of large males required for a particular experiment or study. This is particularly important in large animals where studies involving numerous males are logistically difficult and expensive, and may carry ethical limitations, particularly in primates. However, applications of testis tissue xenografting are limited as manipulation of specific cell types before transplantation is not easily possible and efficiency of spermatogenesis is low in certain donor species14.
The authors have nothing to disclose.
Work from the authors laboratory has been supported by USDA/CSREES/NRICGP (2007-35203-18213); NIH/NCRR (2 R01 RR17359-06), NIH/NIEHS (1 R21 ES014856-01A2) and Alberta Innovates – Health Solutions.
Name of the reagent | Company | Catalogue number |
Collagenase (type IV) | Sigma | C5138 |
Trypsin-EDTA | Invitrogen | 25200-056 |
DNaseI | Sigma | DN25 |
DMEM | Invitrogen | 31053-028 |
Trypan blue stain | Invitrogen | 15250-061 |
Nylon mesh cell strainer | BD biosciences | 352340 (40μm) 352350 (70μm) |
Busulfan | Sigma | B2635 |
Thin-Wall Glass Capillaries | World Precision Instrument | TW 100-3 |
BD intramedic plyethylene tubing (PE100) | BD | CA-63018-725 |
Ethicon 6-0 Silk Suture | Ethicon | 706G |
Wound clips | BD | 427631 |
Sigmacote | Sigma | SL2 |
X-gal | Sigma | B4252 |
Potassium Ferrocyanide | Sigma | P9387 |
Potassium Ferricyanide | Sigma | P3667 |
magnesium chloride | Sigma | 208337 |
sodium deoxycholate | Sigma | D6750 |
N,N-Dimethylformamide | Sigma | D4551 |
Igepal CA-630 | Sigma | 18896 |