Transduction and Transplantation of Spermatogonia into the Testis of Ram Lambs through the Extra‐testicular Rete

Abstract

Spermatogonial transplantation will provide a new way to study spermatogenesis in domestic animals, disseminate male genetics and produce transgenic animals, if efficiency can be improved. We evaluated a 'surgical' method for transplanting donor cells into testes of ram lambs, where the head of the epididymis is reflected, and a catheter introduced into the extra-testicular rete testis. We also tested transduction of ram spermatogonia with a lentiviral (LV) vector as a means to identify permanent colonization, and introduce genes into donor cells. Eight ram lambs, 11- to 13-week olds, were the recipients: in five, spermatogonia were injected into one testis, and the contralateral testis was an un-manipulated control: in two, spermatogonia were injected into one testis and the contralateral was sham-injected: in one, both testes were injected. Six lambs received spermatogonia labelled with a cell-tracking dye and these were collected 1 or 2 weeks after transplantation; three lambs received spermatogonia transduced with a LV vector driving the expression of enhanced Green Fluorescence Protein and these were collected after 2 months. Donor cells were detected by immunohistochemistry in tubules of seven of nine recipient testes. Approximately 22% of tubule cross-sections contained donor cells immediately after transplantation, and 0.2% contained virally transduced cells 2 months after transplantation. The onset of spermatogenesis was delayed, and there were lesions in both injected and sham-injected testes. Despite the effects of the surgery, elongated spermatids were present in one recipient testis 2 months after surgery. The results suggest that, after modifying the surgical and transduction techniques, this approach will be a means to produce good colonization by donor spermatogonia in sheep testes.

Irradiation Enhances the Efficiency of Testicular Germ Cell Transplantation in Sheep

“…The greater availability of stem cell niches in irradiated testes would allow more stem cell colonization. Using the irradiation scheme proposed herein combined with the enrichment of donor cells, the efficiency of the technique could likely be improved [25].…”

“…There are two ways to heighten the efficiency of germ cell transplantation, namely, suitable preparation of the recipient's testis [23] or increased purity of undifferentiated spermatogonia in the donor cell isolation [24,25]. Although donor cells can colonize unprepared recipient testes [10,23], colonization efficiency can be enhanced significantly by providing more stem cell niches in recipient testis for transplanted germ cells [19,24].…”

Transplantation of Autologous Bone Marrow Mesenchymal Stem Cells into the Testes of Infertile Male Rats and New Germ Cell Formation

“…Some studies showed that male adult GCs or spermatogonial stem cells located at the germinal epithelium and formed colonies after transplantation into the testis (39, 40). In the current study, we observed only one possible colony of transplanted cells formed in the testis of the 4-week group (Fig.…”

Recent developments in testis tissue xenografting

“…Germ cell transplantation in the rodent model has proven to be an invaluable tool for the study of spermatogenesis and spermatogonial stem cell biology (Brinster & Avarbock 1994, Brinster & Zimmermann 1994, Brinster 2007. The technique has now also been successfully established in pigs (Honaramooz et al 2002a), goats (Honaramooz et al 2003a, 2003b, 2008b, cattle (Izadyar et al 2003, Herrid et al 2006, monkeys (Schlatt et al 1999(Schlatt et al , 2002a sheep (Rodriguez-Sosa et al 2006, 2009), and dogs (Kim et al 2008; and although its use in these species is now a realistic option, further studies are needed before this approach becomes more widely applicable (Dobrinski & Travis 2007). One current limitation is that germ cell transplantation to study spermatogenesis in these species and to manipulate their germ cells is logistically difficult and expensive.…”