Progress 01/01/08 to 06/30/08
Outputs
OUTPUTS: The long-term goal of the research project is to apply the approach of testis tissue xenografting to the study and manipulation of spermatogenesis in farm animals. SA 1a: To improve generation of haploid gametes by co-grafting of excurrent duct tissue. Fragments of testis tissue from week old Holstein bull calves either with or without parts of the rete testis were collected. Tissue from 7 donors was grafted to 6 mice each. Grafted tissue was collected at 7 mo after transplantation. Animal experiments for this aim have been completed. Analysis of progression of spermatogenesis and assessment of Sertoli cell maturation are ongoing. SA 1b: To support spermatogenic efficiency by modulation of the endocrine environment. In experiment 1, testis tissue fragments from 5 calves were grafted to 12 mice per donor. Six mice per donor were treated with 5 mg/kg acyline every 2 weeks from 3-7 month after grafting to partially suppress testosterone production, and 6 mice served as control. Grafts were collected 7 months after grafting and body weight, graft weight and seminal vesicle weight (indication of testosterone production by the graft tissue) were recorded. While host body weight was unaffected, weights of recovered grafts and of seminal vesicles were lower in treated mice vs controls. Preliminary analysis indicated that percentage of tubules with germ cells was not different but that number of germ cells/100 Sertoli cells was lower in grafts from treated vs control mice. In addition, progression of spermatogenesis appeared slower in treated grafts. Analysis for Sertoli cell maturation is ongoing. A significant effect of donor on graft development was observed In experiment 2, testis tissue from 7 donor calves was grafted to 6 recipient mice each. Half of the recipients were treated with 0.1 % 6-N-propyl-2-thouracil (PTU) for 1 month to induce transient hypothyroidism. Grafts were collected at 5 and 7 months after grafting. Analysis of germ cell number and differentiation and Sertoli cell maturation is ongoing. SA 2: To investigate germ cell developmental potential in cryptorchid testes. Preliminary experiments have been initiated to determine the optimum concentration of equine FSH and LH to support tissue development and germ cell differentiation in equine testis grafts. In addition, control experiments have been performed to further define the effect of donor age and presence of complete spermatogenesis on survival of equine testis grafts. Definitive experiments using cryptorchid equine donors will per performed in the second year of the study. SA 3: To improve spermatogenic efficiency in testis tissue formed de novo from individual cells. Germ cell enrichment by differential plating results in a 3 fold increase in percentage of germ cells in the cell suspension (5% to 15% germ cells) whereas StaPut sedimentation results in a 6-8-fold increase (5% to 30% or 40%). In vivo experiments have been initiated with recipient mice receiving cell pellets from the initial donor cell suspension and from the enriched cell suspension. Resulting de novo formed testis tissue will be collected 8 month after grafting for analysis of spermatogenic efficiency. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Results obtained so far indicate that treatment of host mice to modulate the endocrine environment of the grafted tissue results in detectable effects on donor-derived spermatogenesis. Partial suppression of testosterone production in the grafted tissue by exposing the host mice to a low dose of acyline resulted in lower seminal vesicle and graft weights as expected. When analysis of the grafts for spermatogenic differentiation and Sertoli cell maturation is completed, these experiments will serve as proof of principle that testis tissue xenografting can be employed to test hypotheses and devise strategies to improve male fertility, and to study the effect of substances to enhance or suppress spermatogenesis in large animals without having to perform extensive experiments in the target species. In addition, improved efficiency of de novo generation of testis tissue from isolated cells will provide a novel approach for the study of gene function during spermatogenesis in farm animals.
Publications
- ZENG, W., R. RATHI, H. PAN & I. DOBRINSKI (2007): Comparison of global gene expression between porcine testis tissue xenografts and porcine testis in situ. Mol. Reprod. Dev. 74: 674-679.
- ARREGUI, L., R. RATHI, S.O. MEGEE, A. HONARAMOOZ, M. GOMENDIO, E.R.S. ROLDAN & I. DOBRINSKI (2008): De novo morphogenesis of testis tissue in sheep. 1st World Congress on Reproductive Biology, 54-55 (abstract # 118).
- ARREGUI, L. R. RATHI, W. ZENG, A. HONARAMOOZ, M. GOMENDIO, E.R.S. ROLDAN & I. DOBRINSKI (2008): Xenografting of adult testis tissue. Anim. Reprod. Sci. 106 (1-2): 65-76. (doi:10.1016/j.anireprosci.2007.03.026).
- ARREGUI, L., R. RATHI, S.O. MEGEE, A. HONARAMOOZ, M. GOMENDIO, E.R.S. ROLDAN & I. DOBRINSKI (2008): Xenografting of ovine testis tissue and isolated cells as a model for preservation of material from endangered ungulates. Reproduction 136: 85-93.
- DOBRINSKI, I. & R. RATHI (2008): Ectopic grafting of mammalian testis tissue into mouse hosts. In: HOU, S.X. AND SINGH, S.R. (eds.) Germline Stem Cells. Chapter 10. Methods in Molecular Biology 450, Humana Press, Totowa, NJ; 139-148.
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Progress 01/01/08 to 08/14/07
Outputs
Target Audience:nullChanges/Problems:nullWhat opportunities for training and professional development has the project provided?OUTPUTS: The long-term goal of the research project is to apply the approach of testis tissue xenografting to the study and manipulation of spermatogenesis in farm animals. SA 1a: To improve generation of haploid gametes by co-grafting of excurrent duct tissue. Fragments of testis tissue from week old Holstein bull calves either with or without parts of the rete testis were collected. Tissue from 7 donors was grafted to 6 mice each. Grafted tissue was collected at 7 mo after transplantation. Animal experiments for this aim have been completed. Analysis of progression of spermatogenesis and assessment of Sertoli cell maturation are ongoing. SA 1b: To support spermatogenic efficiency by modulation of the endocrine environment. In experiment 1, testis tissue fragments from 5 calves were grafted to 12 mice per donor. Six mice per donor were treated with 5 mg/kg acyline every 2 weeks from 3-7 month after grafting to partially suppress testosterone production, and 6 mice served as control. Grafts were collected 7 months after grafting and body weight, graft weight and seminal vesicle weight (indication of testosterone production by the graft tissue) were recorded. While host body weight was unaffected, weights of recovered grafts and of seminal vesicles were lower in treated mice vs controls. Preliminary analysis indicated that percentage of tubules with germ cells was not different but that number of germ cells/100 Sertoli cells was lower in grafts from treated vs control mice. In addition, progression of spermatogenesis appeared slower in treated grafts. Analysis for Sertoli cell maturation is ongoing. A significant effect of donor on graft development was observed In experiment 2, testis tissue from 7 donor calves was grafted to 6 recipient mice each. Half of the recipients were treated with 0.1 % 6-N-propyl-2-thouracil (PTU) for 1 month to induce transient hypothyroidism. Grafts were collected at 5 and 7 months after grafting. Analysis of germ cell number and differentiation and Sertoli cell maturation is ongoing. SA 2: To investigate germ cell developmental potential in cryptorchid testes. Preliminary experiments have been initiated to determine the optimum concentration of equine FSH and LH to support tissue development and germ cell differentiation in equine testis grafts. In addition, control experiments have been performed to further define the effect of donor age and presence of complete spermatogenesis on survival of equine testis grafts. Definitive experiments using cryptorchid equine donors will per performed in the second year of the study. SA 3: To improve spermatogenic efficiency in testis tissue formed de novo from individual cells. Germ cell enrichment by differential plating results in a 3 fold increase in percentage of germ cells in the cell suspension (5% to 15% germ cells) whereas StaPut sedimentation results in a 6-8-fold increase (5% to 30% or 40%). In vivo experiments have been initiated with recipient mice receiving cell pellets from the initial donor cell suspension and from the enriched cell suspension. Resulting de novo formed testis tissue will be collected 8 month after grafting for analysis of spermatogenic efficiency. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.How have the results been disseminated to communities of interest?nullWhat do you plan to do during the next reporting period to accomplish the goals?null
Impacts
What was accomplished under these goals?
Results obtained so far indicate that treatment of host mice to modulate the endocrine environment of the grafted tissue results in detectable effects on donor-derived spermatogenesis. Partial suppression of testosterone production in the grafted tissue by exposing the host mice to a low dose of acyline resulted in lower seminal vesicle and graft weights as expected. When analysis of the grafts for spermatogenic differentiation and Sertoli cell maturation is completed, these experiments will serve as proof of principle that testis tissue xenografting can be employed to test hypotheses and devise strategies to improve male fertility, and to study the effect of substances to enhance or suppress spermatogenesis in large animals without having to perform extensive experiments in the target species. In addition, improved efficiency of de novo generation of testis tissue from isolated cells will provide a novel approach for the study of gene function during spermatogenesis in farm animals.
Publications
- Citation:
ZENG, W., R. RATHI, H. PAN & I. DOBRINSKI (2007): Comparison of global gene expression between porcine testis tissue xenografts and porcine testis in situ. Mol. Reprod. Dev. 74: 674-679.
- Citation:
ZENG, W., R. RATHI, H. PAN & I. DOBRINSKI (2007): Comparison of global gene expression between porcine testis tissue xenografts and porcine testis in situ. Mol. Reprod. Dev. 74: 674-679.
Citation:
ARREGUI, L., R. RATHI, S.O. MEGEE, A. HONARAMOOZ, M. GOMENDIO, E.R.S. ROLDAN & I. DOBRINSKI (2008): De novo morphogenesis of testis tissue in sheep. 1st World Congress on Reproductive Biology, 54-55 (abstract # 118).
Citation:
ARREGUI, L. R. RATHI, W. ZENG, A. HONARAMOOZ, M. GOMENDIO, E.R.S. ROLDAN & I. DOBRINSKI (2008): Xenografting of adult testis tissue. Anim. Reprod. Sci. 106 (1-2): 65-76. (doi:10.1016/j.anireprosci.2007.03.026).
- Citation:
ZENG, W., R. RATHI, H. PAN & I. DOBRINSKI (2007): Comparison of global gene expression between porcine testis tissue xenografts and porcine testis in situ. Mol. Reprod. Dev. 74: 674-679.
Citation:
ARREGUI, L., R. RATHI, S.O. MEGEE, A. HONARAMOOZ, M. GOMENDIO, E.R.S. ROLDAN & I. DOBRINSKI (2008): De novo morphogenesis of testis tissue in sheep. 1st World Congress on Reproductive Biology, 54-55 (abstract # 118).
Citation:
ARREGUI, L. R. RATHI, W. ZENG, A. HONARAMOOZ, M. GOMENDIO, E.R.S. ROLDAN & I. DOBRINSKI (2008): Xenografting of adult testis tissue. Anim. Reprod. Sci. 106 (1-2): 65-76. (doi:10.1016/j.anireprosci.2007.03.026).
Citation:
ARREGUI, L., R. RATHI, S.O. MEGEE, A. HONARAMOOZ, M. GOMENDIO, E.R.S. ROLDAN & I. DOBRINSKI (2008): Xenografting of ovine testis tissue and isolated cells as a model for preservation of material from endangered ungulates. Reproduction 136: 85-93.
- Citation:
ZENG, W., R. RATHI, H. PAN & I. DOBRINSKI (2007): Comparison of global gene expression between porcine testis tissue xenografts and porcine testis in situ. Mol. Reprod. Dev. 74: 674-679.
Citation:
ARREGUI, L., R. RATHI, S.O. MEGEE, A. HONARAMOOZ, M. GOMENDIO, E.R.S. ROLDAN & I. DOBRINSKI (2008): De novo morphogenesis of testis tissue in sheep. 1st World Congress on Reproductive Biology, 54-55 (abstract # 118).
Citation:
ARREGUI, L. R. RATHI, W. ZENG, A. HONARAMOOZ, M. GOMENDIO, E.R.S. ROLDAN & I. DOBRINSKI (2008): Xenografting of adult testis tissue. Anim. Reprod. Sci. 106 (1-2): 65-76. (doi:10.1016/j.anireprosci.2007.03.026).
Citation:
ARREGUI, L., R. RATHI, S.O. MEGEE, A. HONARAMOOZ, M. GOMENDIO, E.R.S. ROLDAN & I. DOBRINSKI (2008): Xenografting of ovine testis tissue and isolated cells as a model for preservation of material from endangered ungulates. Reproduction 136: 85-93.
Citation:
DOBRINSKI, I. & R. RATHI (2008): Ectopic grafting of mammalian testis tissue into mouse hosts. In: HOU, S.X. AND SINGH, S.R. (eds.) Germline Stem Cells. Chapter 10. Methods in Molecular Biology 450, Humana Press, Totowa, NJ; 139-148.
- Citation:
ZENG, W., R. RATHI, H. PAN & I. DOBRINSKI (2007): Comparison of global gene expression between porcine testis tissue xenografts and porcine testis in situ. Mol. Reprod. Dev. 74: 674-679.
Citation:
ARREGUI, L., R. RATHI, S.O. MEGEE, A. HONARAMOOZ, M. GOMENDIO, E.R.S. ROLDAN & I. DOBRINSKI (2008): De novo morphogenesis of testis tissue in sheep. 1st World Congress on Reproductive Biology, 54-55 (abstract # 118).
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Progress 01/01/05 to 12/31/05
Outputs
The overall objective of this project is to develop a novel approach for the study and manipulation of spermatogenesis in farm animals through testis tissue xenotransplantation. We demonstrated that spermatogenesis can proceed to the stage of haploid gametes in testis tissue xenografts from cattle and horses. However, time to sperm production was not shortened as we had previously reported for primate testis xenografts, and spermatogenic efficiency was low in testis xenografts from cattle and horses compared to small ruminants and pigs. In the equine , we found that spermatogenic differentiation on the mouse host depended upon the maturity of the tissue at the time of grafting. For immature donor testis tissue spermatogenic differentiation following grafting varied from no differentiation to the appearance of haploid cells. For mature donor testis samples, following grafting an initial loss of differentiated germ cells was observed prior to a resurgence of
spermatogenesis. These results indicate that horse spermatogenesis can occur in a mouse host albeit with low efficiency. Supplementation of exogenous gonadotropins to mice carrying bovine testis xenografts did not improve spermatogenic efficiency. While the mean weight of seminal vesicles in the host mice was comparable to that observed in intact male mice, some grafts grew very big and the hosts had very large seminal vesicles, indicating the possibility that feedback to the mouse pituitary was inefficient and too much testosterone was produced by the xenografts. In contrast, administration of exogenous gonadotropins did appear to support post-meiotic differentiation in horse testis grafts. Administration of exogenous growth hormone (rbST) to mice carrying bovine grafts did not significantly improve spermatogenic efficiency. We investigated whether testis tissue can be preserved by cooling, conventional freezing or vitrification and if sperm recovered from xenografts can maintain
viability after freezing and thawing. Piglet testis tissue was cryopreserved with 3 vitrification protocols and a conventional cell freezing protocol. Post-thaw viability was significantly higher with one vitrification protocol and conventional freezing than with the other protocols. The highest post-thaw cell viability was observed after vitrification with the lowest concentration of cryoprotectant. Germ cell viability was high for conventional freezing and vitrification. Complete spermatogenesis was observed in grafts from all preservation treatments with levels comparable to fresh grafts in tissue kept in cold storage and lower rates of differentiation for frozen thawed tissue. Overall, the results indicated that developmental potential of testis tissue is preserved by cooling, conventional freezing or vitrification and that sperm recovered from xenografts can be stored frozen for future use in assisted reproduction.
Impacts
Production of sperm from the testes of immature domestic animals provides a novel way to study and manipulate male fertility in economically important species. The system will allow us to generate sperm from valuable immature animals, and to study the effects of drugs to enhance or suppress spermatogenesis without having to perform experiments in the target species.
Publications
- RATHI, R, A. HONARAMOOZ, W. ZENG, S. SCHLATT, & I. DOBRINSKI (2005): Germ cell fate in bovine testis tissue xenografts. XXVIII North American Testis Workshop, 113 (abstract 22).
- TURNER, R.M., R. RATHI, W. ZENG & I. DOBRINSKI (2005): Xenografting of degenerate stallion testis onto a mouse host does not rescue the testicular degeneration phenotype. Anim. Reprod. Sci. 89 (1-4): 253-255.
- TURNER, R., R. RATHI, A. HONARAMOOZ, W. ZENG, & I. DOBRINSKI (2005): Germ cell development in equine testis tissue xenografted into mouse hosts. Reprod. Fertil. Dev.17 (1,2): 283 (abstract 268).
- RATHI, R. A. HONARAMOOZ, W. ZENG, R. TURNER & I. DOBRINSKI (2005): Testis tissue xenografting as bioassay for germ cell developmental potential in equine cryptorchid testes. Reprod. Fertil. Dev.17 (1,2): 283 (abstract 267).
- HONARAMOOZ, A., W. ZENG, R. RATHI, J. KOSTER, O. RYDER, & I. DOBRINSKI (2005): Testis tissue xenografting to preserve germ cells from a cloned banteng calf. Reprod. Fertil. Dev.17 (1,2): 247 (abstract 193).
- RATHI, R., A. HONARAMOOZ, W. ZENG, S. SCHLATT & I. DOBRINSKI (2005): Germ cell fate and seminiferous tubule development in bovine testis xenografts. Reproduction 130; 923-929.
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Progress 01/01/04 to 12/31/04
Outputs
The overall objective of this grant is to develop a novel approach for the study and manipulation of spermatogenesis in farm animals through testis tissue xenotransplantation. In 2004 we have completed specific aim 1a and further addressed specific aim 1b, 1c and 1d. We also initiated experiments directed at specific aim 2b. Results pertinent to specific aim 1a (To extend the technique of testis tissue xenografting to slow maturing large domestic animals using cattle and horses as models). Fragments of testis from 1 wk old calves were grafted under the back skin of immunodeficient castrated male mice. Grafts were recovered at 1.5 to 10 mo post grafting. Between 6 and 7 mo spermatogenesis seemed to be arrested in most tubules in the bovine grafts with loss of post meiotic germ cells. The results indicated that low efficiency of sperm production is due to an initial loss of germ cells and impaired germ cell survival during meiotic and post-meiotic differentiation in
bovine testis xenografts. To investigate the applicability of xenografting to horse testis tissue, fragments of testis tissue from 4 sexually immature colts were grafted under the back skin of mice. Histological examination was performed between 14 and 50 wk post-transplantation. Pachytene spermatocytes were observed in testis grafts from the 5 and 8 mo old donors from 14 wk onward. Spermatogenesis did not proceed through meiosis in grafts from the 5 mo old donor. The results indicated that horse spermatogenesis can occur in a mouse host albeit with low efficiency. To explore, whether cryptorchid horse testes contained germ cells capable of differentiation, fragments of abdominally cryptorchid testis tissue from 3 donor horses were grafted under the back skin of mice. Histological examination was performed between 5 and 45 weeks post-transplantation. By 28 wks after grafting, pachytene spermatocytes were observed in xenografts from all cryptorchid donor testes. The results indicated
that even after 3 years of exposure to core body temperature, equine cryptorchid testes contain germ cells capable of differentiation. Treatment of recipient mice with exogenous gonadotropins or rBST did not result in significant acceleration of germ cell differentiation. We investigated whether testis tissue can be preserved by vitrification. Piglet testis tissue was cryopreserved with 3 vitrification protocols and a conventional freezing protocol. The results demonstrated that the concentration of penetrating cryoprotectants can be reduced to as little as 20 percent and still allow effective cryopreservation of pig testis tissue. Testis tissue preserved with these vitrification protocols has been grafted into host mice and graft development will be monitored. Isolated testis cells were grafted under the back skin of recipient mice and the re-establishment of tubular structures and germ cell differentiation was monitored over time. Experiments are still ongoing. In summary, the
experiments performed during 2004 were productive and have provided the basis for future work aimed at the study and manipulation of testis function in farm animals through testis tissue xenografting.
Impacts
Production of sperm from the testes of immature domestic animals provides a novel way to study and manipulate male fertility in economically important species. The system will allow us to generate sperm from valuable immature animals, shorten the generation interval and thereby accelerate genetic progress. It will also allow us to study the effects of drugs to enhance or suppress spermatogenesis without having to perform experiments in the target species.
Publications
- SNEDAKER, A., A. HONARAMOOZ & I. DOBRINSKI (2004): A game of cat and mouse: Xenografting of testis tissue from domestic kittens results in complete cat spermatogenesis in a mouse host. J. Androl. 25(6); 926-930.
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Progress 01/01/03 to 12/31/03
Outputs
The overall objective of this grant was to develop a novel approach for the study and manipulation of spermatogenesis in farm animals through testis tissue xenotransplantation. Testis tissue from 1-week-old Holstein calves was grafted under the back skin of immunodeficient recipient mice. By 20 weeks after grafting, germ cells in the grafts had entered meiosis and pachytene spermatocytes were evident. By 31 weeks after grafting, haploid spermatids were seen in the tubules and more than 41 weeks after grafting, mature spermatozoa could be found. To explore the horse as donor species, testis tissue was grafted from immature donors ranging from 2 weeks to 10 months of age. Meiotic germ cells (pachytene spermatocytes) were observed by 18-25 weeks after grafting and sperm heads by 35 weeks after grafting. Taking into account differences in donor age, the time course of development of grafted testis tissue was similar to that observed in the bovine. However, tissue from a
2-week-old horse did not develop, which is in contrast to our findings in the bovine. In addition to grafting of scrotal testis tissue, experiments were also directed at using cryptorchid testes as donor tissue. We could demonstrate that xenografting served to initiate spermatogenesis in a previously cryptorchid testis recovered from a yearling horse. Mice carrying bovine testis xenografts were treated with PMSG using osmotic minipumps or a long-acting formulation of rBST. Results were variable and no clear effect on timing of testicular maturation could be demonstrated. Future experiments using different treatment protocols will investigate this option further. Mice carrying horse testis xenografts were treated with PMSG and hCG. Results were not significant but there was a tendency for earlier development with pachytene spermatocytes observed at 14 weeks after grafting compared to 18-25 weeks in controls. We investigated whether testis tissue can be preserved by vitrification.
Piglet testis tissue was cryopreserved with 3 vitrification protocols and a conventional freezing protocol. Post-thaw viability was significantly higher with the first vitrification protocol and conventional freezing than with the other vitrification protocols. The highest post-thaw cell viability was observed after vitrification with the lowest concentration of cryoprotectant. These results demonstrate that the concentration of penetrating cryoprotectants can be reduced to as little as 20 percent and still allow effective cryopreservation of pig testis tissue. Testis tissue preserved with these vitrification protocols has been grafted into host mice and graft development will be monitored. Recipient mice producing xenogeneic sperm have been generated. Initial experiments were directed at optimizing oocyte maturation protocols and ICSI technique. Ongoing experiments are aimed at generating live offspring following ICSI and ET.
Impacts
Our first months of study were productive and have provided the basis for future work aimed at the study and manipulation of testis function in farm animals through testis tissue xenografting.
Publications
- No publications reported this period
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