Germ Cell Transplantation and Testis Tissue Xenografting in Mice

Summary

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.

Abstract

Germ cell transplantation was developed by Dr. Ralph Brinster and colleagues at the University of Pennsylvania in 1994^1,2. 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 transplantation^4,5, to study Sertoli cell-germ cell interaction^6,7, SSC homing and colonization^3,8, as well as SSC self-renewal and differentiation^9,10.

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.

Protocol

PART A. Germ cell transplantation in mice

1. Preparation of recipient mice

1. Recipients should be immunologically tolerant (either genetically matched to donors or immune-deficient) to the donor testis cells.
2. Recipients should be either naturally devoid of spermatogenesis (e.g. W/ W^v mice) or depleted of endogenous germ cells. Germ cell depletion can be achieved by irradiation or chemotherapeutic drugs such as Busulfan. In this protocol, we describe the method of preparing recipient mice with Busulfan.
3. Treat recipients at 4-6 week of age through i.p injection of Busulfan. The optimal dose of B....

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Collagenase (type IV)	Sigma-Aldrich	C5138
Trypsin-EDTA	Invitrogen	25200-056
DNaseI	Sigma-Aldrich	DN25
DMEM	Invitrogen	31053-028
Trypan blue stain	Invitrogen	15250-061
Nylon mesh cell strainer	BD Biosciences	352340 (40µm) 352350 (70µm)
Busulfan	Sigma-Aldrich	B2635
Thin-Wall Glass Capillaries	World Precision Instruments, Inc.	TW 100-3
BD intramedic plyethylene tubing (PE100)	BD Biosciences	CA-63018-725
Ethicon 6-0 Silk Suture	Ethicon Inc.	706G
Wound clips	BD Biosciences	427631
Sigmacote	Sigma-Aldrich	SL2
X-gal	Sigma-Aldrich	B4252
Potassium Ferrocyanide	Sigma-Aldrich	P9387
Potassium Ferricyanide	Sigma-Aldrich	P3667
magnesium chloride	Sigma-Aldrich	208337
sodium deoxycholate	Sigma-Aldrich	D6750
N,N-Dimethylformamide	Sigma-Aldrich	D4551
Igepal CA-630	Sigma-Aldrich	18896