Rahul Rathi

Preservation and transplantation of porcine testis tissue.

Grafting of immature mammalian testis tissue to mouse hosts can preserve the male germline. To make this approach applicable to a clinical or field situation, it is imperative that the testis tissue and/or spermatozoa harvested from grafted tissue are preserved successfully. The aim of the present study was to evaluate protocols for the preservation of testis tissue in a porcine model. Testis tissue was stored at 4 degrees C for short-term preservation or cryopreserved by slow-freezing, automated slow-freezing or vitrification for long-term storage. Preserved tissue was transplanted ectopically to mouse hosts and recovered xenografts were analysed histologically. In addition, spermatozoa were harvested from xenografts and cryopreserved. Total cell viability and germ cell viability remained high after tissue preservation. Complete spermatogenesis occurred in xenografts preserved by cooling up to 48 h, whereas spermatogenesis progressed to round spermatids in the xenografts that were frozen-thawed before grafting. Approximately 50% of spermatozoa harvested from xenografts remained viable after freezing and thawing. The in vivo developmental potential of cryopreserved tissue was reduced despite high post-thaw viability. Therefore, it is important to evaluate germ cell differentiation in vivo in addition to cell viability in vitro when optimising freezing protocols for testis tissue.

Building a Testis: Formation of Functional Testis Tissue after Transplantation of Isolated Porcine (Sus scrofa) Testis Cells

Abstract During mammalian development, morphogenesis of the testis requires the coordinated interplay of somatic cells to form seminiferous cords in which the primitive germ cells reside. These cords are the precursor of the functional male gonad and as such form the basis of male fertility. Cell migration during mammalian organogenesis and formation of complex tissues, such as the testis, are difficult to study in situ. Herein, we report extensive rearrangement of cells to regenerate complete functional testis tissue after implantation of isolated neonatal porcine testis cells under the skin of immunodeficient mice. Somatic cells and germ cells reorganized into structures that have remarkable morphologic and physiologic similarity to normal testis tissue, forming the endocrine and spermatogenic compartment of the testis. This unique in vivo system provides an accessible model for the study of testicular morphogenesis that could be especially useful in nonrodent species.

Xenografting of sheep testis tissue and isolated cells as a model for preservation of genetic material from endangered ungulates

Recovery of germ cells could be an option for preservation of the genetic pool of endangered animals. In immature males, xenografting of testis tissue provides the opportunity to recover sperm from these animals. In adult animals, xenografting has been less successful, but de novo morphogenesis of functional testis tissue from dissociated testis cells could be an alternative. To assess the potential use of these techniques in endangered bovid species, the domestic sheep was used as a model. Testes from 2-week-old lambs were grafted as tissue fragments or cell suspensions into nude mice. Grafts were recovered at 4, 8, 12 and 16 weeks post grafting. For isolated cells, two additional time points at 35 and 40 weeks after grafting were added. In addition, to analyse the possible effect of social stress among mice within a group on the development of the grafts, testis tissue grafts were recovered 13 weeks post grafting from mice housed individually and in groups. Complete spermatogenesis occurred in sheep testis xenografts at 12 weeks, similar to the situation in situ. Isolated sheep testis cells were able to reorganize and form functional testicular tissue de novo. Housing mice individually or in groups did not have any effect on the development of xenografts. Xenografting of testis tissue might be useful to obtain sperm from immature endangered ungulates that die prematurely. Testis tissue de novo morphogenesis from isolated cells could open interesting options to recover germ cells from mature males with impaired spermatogenesis.

Maturation of Testicular Tissue from Infant Monkeys after Xenografting into Mice

In juvenile monkeys, precocious puberty can be induced by administration of gonadotropins resulting in testicular somatic cell maturation and germ cell differentiation. It is, however, unknown whether testicular maturation can also be induced in younger monkeys. Here we used testis tissue xenografting to investigate whether infant monkey testis tissue will undergo somatic cell maturation and/or spermatogenesis in response to endogenous adult mouse gonadotropins or exogenous gonadotropins. Testicular tissue pieces from 3- and 6-month-old rhesus monkeys were grafted to immunodeficient, castrated mice. Recipient mice were either left untreated or treated with pregnant mare serum gonadotropin and/or human chorionic gonadotropin twice weekly and were killed 28 weeks after grafting. Testicular maturation in grafted tissue was assessed based on morphology and the most advanced germ cell type present and by immunohistochemistry for expression of proliferating cell nuclear antigen, Mullerian-inhibiting substance, and androgen receptor. Testis grafts, irrespective of donor age or treatment, contained fewer germ cells than donor tissue. Grafts from 6-month-old donors showed tubular expansion with increased seminiferous tubule diameter and lumen formation, whereas those harvested from gonadotropin-treated mice contained elongated spermatids. Grafts from 3-month-old donors recovered from gonadotropin-treated mice contained pachytene spermatocytes, whereas those recovered from untreated mice showed only slight tubular expansion. Immunohistochemistry revealed that exposure to exogenous gonadotropins supported Sertoli cell maturation, irrespective of donor age. These results indicate that sustained gonadotropin stimulation of immature (<12 months old) monkey testis supports Sertoli cell maturation, thereby terminating the unresponsive phase of the germinal epithelium and allowing complete spermatogenesis in testis tissue from infant rhesus monkeys.

Lymphoid-Specific Helicase (HELLS) Is Essential for Meiotic Progression in Mouse Spermatocytes

Lymphoid-specific helicase (HELLS; also known as LSH) is a member of the SNF2 family of chromatin remodeling proteins. Because Hells-null mice die at birth, a phenotype in male meiosis cannot be studied in these animals. Allografting of testis tissue from Hells−/− to wild-type mice was employed to study postnatal germ cell differentiation. Testes harvested at Day 18.5 of gestation from Hells−/−, Hells+/−, and Hells+/+ mice were grafted ectopically to immunodeficient mice. Bromodeoxyuridine incorporation at 1 wk postgrafting revealed fewer dividing germ cells in grafts from Hells−/− than from Hells+/+ mice. Whereas spermatogenesis proceeded through meiosis with round spermatids in grafts from Hells heterozygote and wild-type donor testes, spermatogenesis arrested at stage IV, and midpachytene spermatocytes were the most advanced germ cell type in grafts from Hells−/− mice at 4, 6, and 8 wk after grafting. Analysis of meiotic configurations at 22 days posttransplantation revealed an increase in Hells−/− spermatocytes with abnormal chromosome synapsis. These results indicate that in the absence of HELLS, proliferation of spermatogonia is reduced and germ cell differentiation arrested at the midpachytene stage, implicating an essential role for HELLS during male meiosis. This study highlights the utility of testis tissue grafting to study spermatogenesis in animal models that cannot reach sexual maturity.

Development of Bovine Fetal Testis Tissue After Ectopic Xenografting in Mice

Testis tissue xenografting represents a versatile model to study testis biology, and to preserve fertility in immature animals. To evaluate whether bovine fetal testes can mature when grafted into mouse hosts, small fragments of testes from midgestation (125 to 145 days of gestation) bovine fetuses were grafted ectopically into immunodeficient castrated male mice. At grafting, donor tissue displayed the typical seminiferous cords composed of gonocytes and primitive Sertoli cells. At 5 or 10 months after grafting, weight of the seminal vesicles in recipient mice was indicative of production of bioactive testosterone by xenografts. Xenografts showed similar development regardless of donor age. At 5 months, tubule formation occurred but germ cell differentiation had not proceeded beyond the spermatogonia stage. At 10 months, an increase in tubule size was evident and pachytene spermatocytes were observed as the most advanced type of germ cells in the xenografts of 2 donors. The number of tubules with germ cells was reduced in xenografts compared to donor tissue, but at 10 months the number of germ cells per tubule was higher than in donors. Germ cell proliferation was similar in donor tissue and xenografts. However, Sertoli cells showed a higher proliferation rate in xenografts collected at 5 months than in donor fetal testes and xenografts collected at 10 months. Sertoli cells in xenografts showed a progressive but incomplete loss of expression of Müllerian inhibiting substance and weak androgen receptor expression, indicating an incomplete Sertoli cell maturation. In conclusion, fetal testis tissue developed partially, qualitatively similar to pubertal testes in situ.

Xenografting restores spermatogenesis to cryptorchid testicular tissue but does not rescue the phenotype of idiopathic testicular degeneration in the horse (Equus caballus)

Spermatogenesis from many mammalian species occurs in fragments of normal testis tissue xenografted to mice. Here we apply xenografting to the study of testicular pathology. Using the horse model, we investigated whether exposure to a permissive extratesticular environment in the mouse host would rescue spermatogenesis in cryptorchid testicular tissue or in tissue affected by idiopathic testicular degeneration (ITD). In cryptorchid tissue, where the extratesticular environment is abnormal, xenografting induced spermatogenesis up to meiosis in a subpopulation of seminiferous tubules. Thus, spermatogonia survive and partially retain their potential to differentiate in cryptorchid horse testes. In contrast, the primary defect in equine ITD is hypothesised to be tissue autologous. In support of this, xenografting did not restore spermatogenesis to tissue affected by ITD, thus confirming that the testis itself is primarily diseased. This outcome was not affected by supplementation of exogenous gonadotropins to the mouse host or by reconstitution of a normal reproductive regulatory axis supplied by functional porcine testicular xenografts. These studies demonstrate the usefulness of xenografting for the study of testicular pathology.

Ectopic Grafting of Mammalian Testis Tissue into Mouse Hosts

Mammalian spermatogenesis is a highly organized process of cell division and differentiation that requires intimate contact between germ cells and testicular somatic cells. Lack of a suitable in vitro system has caused many aspects of spermatogenesis, especially in nonrodent species, to remain elusive. We describe ectopic grafting of testis tissue from sexually immature males to immunodeficient mouse hosts as an in vivo culture system that allows recapitulation of complete spermatogenesis from diverse mammalian species with the production of fertilization-competent sperm in a mouse host. In this system, the donor species testicular environment is preserved allowing experimentation in a small rodent. The accessibility of the tissue in the mouse host makes it possible to manipulate spermatogenesis and steroidogenesis in a controlled manner that is often not feasible in the donor species. It also allows detailed analysis of the effects of toxins and compounds to enhance or suppress male fertility in an in vivo system without extensive experimentation in the target species. Finally, as it provides a source of male gametes even from immature gonads, grafting of fresh or preserved testis tissue offers an invaluable tool for the conservation of fertility in males if sperm cannot be obtained for cryopreservation.

Endocrine modulation of the recipient environment affects development of bovine testis tissue ectopically grafted in mice

Testis tissue xenografting is a powerful approach for the study of testis development and spermatogenesis, and for fertility preservation in immature individuals. In bovine testis xenografts, maturation and spermatogenesis are inefficient when compared to other species. To evaluate if exogenous modulation of the endocrine milieu in recipient mice will affect spermatogenic efficiency in xenografts from newborn calves, recipient mice were treated with the GnRH antagonist acyline (5 mg/kg s.c. every 2 weeks) to reduce testosterone production in xenografts, or with 6-N-propyl-2-thiouracil (PTU, 0.1% in drinking water for 4 weeks), to induce transient hypothyroidism in recipient mice respectively. Both treatments altered developmental parameters of testis xenografts and reduced germ cell differentiation. While the effects of acyline treatment can be attributed to inhibition of GnRH and gonadotropin action, lower Sertoli cell numbers and decreased seminiferous tubule length observed after PTU treatment were opposite to effects reported previously in rats. Regardless of treatment, Sertoli cells underwent only partial maturation in xenografts as Müllerian inhibiting substance and androgen receptor expression were lower than in donor and adult tissue controls respectively. In conclusion, although treatments did not result in improvement of maturation of bovine testis xenografts, the current study demonstrates that exogenous modulation of the endocrine milieu to affect xenograft development in recipient mice provides an accessible model to study endocrine control of spermatogenesis in large donor species.

Suppression of spermatogenesis before grafting increases survival and supports resurgence of spermatogenesis in adult mouse testis

OBJECTIVE To test whether absence of complete spermatogenesis in mature testicular tissue before grafting will increase graft survival. DESIGN Prospective experimental study. SETTING Laboratory. ANIMAL(S) Donor testes were obtained from adult untreated mice, adult mice rendered cryptorchid, and adult mice treated with a GnRH antagonist (acyline). INTERVENTION(S) Donor testes were ectopically grafted to nude mice and recovered at three time points. MAIN OUTCOME MEASURE(S) Most advanced germ cell type and presence of spermatogonia were assessed. Donor testes and grafts were analyzed by histology and by immunocytochemistry for ubiquitin C-terminal hydrolase-L1 to mark germ cells. RESULT(S) Suppression of spermatogenesis by inducing cryptorchidism or acyline treatment resulted in improved survival of grafted tissue compared with controls and recovery of complete spermatogenesis, whereas control testis grafts mostly degenerated and did not restore complete spermatogenesis. CONCLUSION(S) These results indicate that complete spermatogenesis at the time of grafting has a negative effect on graft survival. Grafting of adult testis tissue from donors with suppressed spermatogenesis leads to spermatogenic recovery and may provide a tool to study and preserve fertility and for conservation of genetic resources in individuals that lack complete germ cell differentiation.