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Donor-host involvement in immature rat testis xenografting into nude mouse hosts.

ABSTRACT: Immature testicular tissue of a wide variety of mammalian species continues growth and maturation when ectopically grafted under the dorsal skin of adult nude mouse recipients. Tissues from most donor species fully mature, exhibiting complete spermatogenesis within months. The connection to the recipient's vascular system is mandatory for graft development, and failure of vascularization leads to necrosis in the grafted tissue. In the present study, we analyze to what extent 1) the xenografted immature donor tissue and 2) the recipient's cells and tissues contribute to the functional recovery of a "testicular xenograft." We address whether recipient cells migrate into the testicular parenchyma and whether the circulatory connection between the donor testicular tissue and the recipient is established by ingrowing host or outgrowing donor blood vessels. Although this issue has been repeatedly discussed in previous xenografting studies, so far it has not been possible to unequivocally distinguish between donor and recipient tissues and thus to identify the mechanisms by which the circulatory connection is established. To facilitate the distinction of donor and recipient tissues, herein we used immature green fluorescent protein-positive rat testes as donor tissues and adult nude mice as graft recipients. At the time of graft recovery, donor tissues could be easily identified by the GFP expression in these tissues, allowing us to distinguish donor- and recipient-derived blood vessels. We conclude that the circulatory connection between graft and host is established by a combination of outgrowing small capillaries from the donor tissue and formation of larger vessels by the host, which connect the graft to subcutaneous blood vessels.

SUBMITTER: Schlatt S

PROVIDER: S-EPMC2857632 | biostudies-literature | 2010 May

REPOSITORIES: biostudies-literature

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Donor-host involvement in immature rat testis xenografting into nude mouse hosts.

Schlatt Stefan S Westernströer Birgit B Gassei Kathrin K Ehmcke Jens J

Biology of reproduction 20100127 5

Immature testicular tissue of a wide variety of mammalian species continues growth and maturation when ectopically grafted under the dorsal skin of adult nude mouse recipients. Tissues from most donor species fully mature, exhibiting complete spermatogenesis within months. The connection to the recipient's vascular system is mandatory for graft development, and failure of vascularization leads to necrosis in the grafted tissue. In the present study, we analyze to what extent 1) the xenografted i ...[more]

PMID: 20107205

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Project description:The study of spermatogenesis in the horse is challenging because of the absence of an in vitro system that is capable of reproducing efficient spermatogenesis and because of the difficulties and costs associated with performing well-controlled studies in vivo. In an attempt to develop novel methods for the study of equine spermatogenesis, we tested whether cells from enzymatically digested pre-pubertal equine testicular tissue were capable of de novo tissue formation and spermatogenesis following xenografting under the back skin of immunocompromised mice. Testes were obtained from normal pre-pubertal colts and dissociated into cell suspensions using trypsin/collagenase digestion. Resulting cell pellets, consisting of both somatic and germ cells, were injected into fascial pockets under the back skin of immunocompromised, castrated mice and maintained for between 1 and 14 months. Mice were killed and grafts were recovered and analyzed. As has been reported for testis cell suspensions from pigs, mice, cattle, and sheep, de novo formation of equine testicular tissue was observed, as evidenced by the presence of seminiferous tubules and an interstitial compartment. There was an increased likelihood of de novo testicular formation as grafting period increased. Using indirect immunofluorescence, we confirmed the presence of spermatogonia in de novo formed seminiferous tubules. However, we found no evidence of meiotic or haploid cells. These results indicate that dissociated pre-pubertal equine testis cells are capable of reorganizing into the highly specialized endocrine and spermatogenic compartments of the testis following ectopic xenografting. However, in spite of the presence of spermatogonia within the seminiferous tubules, spermatogenesis does not occur. Although this technique does allow access to the cells within the seminiferous tubule and interstitial compartments of the equine testis prior to reaggregation, the absence of spermatogenesis will limit its use as a method for the study of testicular function in the horse.

Project description:BackgroundCurrently there are no established fertility preservation options for pre-pubertal boys facing cancer treatment. Granulocyte-colony stimulating factor (G-CSF) treatment has been proposed to be chemoprotective against spermatogonial cell loss in an alkylating chemotherapy model of busulfan treated adult mice. Having previously shown that exposure to the alkylating-like chemotherapy cisplatin resulted in a reduction in germ cell numbers in immature human testicular tissues, we here investigate whether G-CSF would prevent cisplatin-induced germ cell loss in immature human and mouse (fetal and pre-pubertal) testicular tissues.MethodsOrganotypic in vitro culture systems were utilised to determine the effects of clinically-relevant concentrations of G-CSF in cisplatin-exposed immature testicular tissues. Human fetal (n = 14 fetuses) and mouse pre-pubertal (n = 4 litters) testicular tissue pieces were cultured and exposed to cisplatin or vehicle control for 24 hrs and analysed at 72 and 240 hrs post-exposure. Combined G-CSF and cisplatin exposure groups explored varying concentrations and duration of G-CSF supplementation to the culture medium (including groups receiving G-CSF before, during and after cisplatin exposure). In addition, effects of G-CSF supplementation alone were investigated. Survival of total germ cell and sub-populations were identified by expression of AP2γ and MAGE-A4 for human gonocytes and (pre)spermatogonia, respectively, and MVH and PLZF, for mouse germ cells and putative spermatogonial stem cells (SSCs) respectively, were quantified.ResultsExposure to cisplatin resulted in a reduced germ cell number in human fetal and mouse pre-pubertal testicular tissues at 240 hrs post-exposure. Germ cell number was not preserved by combined exposure with G-CSF using any of the exposure regimens (prior to, during or after cisplatin exposure). Continuous supplementation with G-CSF alone for 14 days did not change the germ cell composition in either human or mouse immature testicular tissues.ConclusionsThis study demonstrates that exposure to G-CSF does not prevent cisplatin-induced germ cell loss in immature human and mouse testicular tissues in an in vitro system.

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Donor-host involvement in immature rat testis xenografting into nude mouse hosts.

Publications

Donor-host involvement in immature rat testis xenografting into nude mouse hosts.

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