Progress 09/01/01 to 08/31/04
Outputs
The overall objective of this project was to develop a completely in vitro culture system for bovine spermatogenesis. After promising early results, the major limitation has been maintaining a viable germ cell population in vitro. In an effort to overcome this limitation, we attempted to develop an immortalized spermatogonial cell line by transfecting dissociated bovine testis cells with the human reverse transcriptase component of telomerase (hTERT. Testes from bulls ranging from 8 to 32 weeks of age were enzymatically dissociated into seminiferous tubule fragments and transduced in vitro using a gibbon ape leukemia retrovirus (GALV) expressing retroviral vector pBMN-GFP. In vitro-cultured bovine testis cells were immortalized by stable tranduction of the catalytic subunit of telomerase reverse transcriptase (hTERT) via a GALV. Testis cells that stably integrated the hTERT gene were selected with puromycin. Introduction of hTERT was confirmed by RT-PCR in testis cells
from the range of bull ages tested. Bovine testis cell lines expressing hTERT were characterized by RT-PCR with Sertoli (SCF), peritubular cell (LIF) and germ-cell (vasa) specific primers. The 8 week testis cells with hTERT expressed SCF and LIF but not vasa demonstrating that the immortalized testis cells cells include Sertoli and peritubular cell populations but not germ cells. Further characterization by RT-PCR for expression of additional Sertoli cell genes such as FSH receptor and inhibin is planned. Because of the persistent difficulty in maintaining seminiferous tubule architecture and characterizing germ cell development in the bull testis in vitro, a study to characterize germ cell progression during testis development in vivo and correlate germ cell progression with stage-specific gene expression was undertaken. Holstein bulls were castrated as neonates and at 8, 12, and 16 weeks of age and at 2 week intervals from 16-32 weeks of age (3 bulls per age group). Blood for
testosterone determination was collected by jugular puncture and scrotal circumference was measured weekly from 8-32 weeks for the oldest age group and at the time of castration for other age groups. Testes were weighed at castration and samples of parenchyma were fixed for histological processing, PAS staining, and thick sectioning; placed in culture medium for in vitro culture; grafted subcutaneously into nude mice; or frozen in liquid nitrogen for RNA extraction. Progression of spermatogenesis in vivo and in grafts after 75 and 175 days and expression of c-kit, TH2B, TP1, protamine 2, vasa, and GFRa (germ cells); and LIF (peritubular), SCF and FSH receptor (Sertoli), and P450scc (Leydig cells) are being evaluated currently. Effect of developmental age of the testis on spermatogenesis in vitro was also evaluated based on morphological criteria and germ cell apoptosis. However, organization of the seminiferous tubules was lost within1-2 weeks in vitro regardless of the age of the
bull. The data from this study should contribute to efforts toward an improved understanding of spermatogenic progression in the bull and procedures for developing and assessing procedures for spermatogenesis in vitro.
Impacts
A completely in vitro culture system for spermatogenesis will provide a basis for mechanistic studies of spermatogenesis, a model for studying and manipulating spermatogenesis in vitro and in vivo, the potential for genetic modification of the male germ line, and an approach to treating selected causes of male-factor infertility.
Publications
- Bartholomew, R.A. and Parks, J.E. 2005. Identification, cloning, and localization of VASA homolog in fetal bovine gonads. Biol. Reprod. in preparation.
- Parks, J.E. 2004. Spermatogenesis, sperm transport, and semen. Encyc. Anim. Sci. pp. 810-813, Marcel Dekker, Inc. New York, NY.
- Parks, J.E. and Bartholomew, R.A. 2004. Expression and localization of VASA and Oct-4 in fetal bovine germ cells. Prog. 37th Ann. Mtg. Soc. Study Reprod., p. 128.
- Bartholomew, R.A. and Parks, J.E. 2002. The cloning of bovine VASA in fetal gonads. Biol. Reprod. 66(Suppl. 1):161
- Kaproth, M.T., Lee D.R. and Parks, J.E. 2002. Transplantation of testicular explants from prepubertal bulls to nude mice and ex situ production of haploid germ cells over a 20-week period. J. Dairy Sci. 85(Suppl.1):302.
- Lee, D. R. and Parks, J. E. 2001. Embryo production by intracytoplasmic injection of haploid germ cells produced in vitro from neonatal bull testis. Biol. Reprod. 64 (Suppl. 1):131.
- Lee, D. R., Kaproth, M. T. and Parks, J. E. 2001. In vitro production of haploid germ cells from fresh or frozen-thawed testicular cells of neonatal bulls. Biol. Reprod. 65:834-839
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Progress 01/01/03 to 12/31/03
Outputs
The overall objective of this project is to develop a completely in vitro culture system for mammalian spermatogenesis using the bull as a model. Previously, neonatal calf testes were dissociated enzymatically, reaggregated using phytohemagglutinin, encapsulated in alginate, and cultured at 32 degrees C. Stage-specific gene expression and ploidy analysis indicated that spermatogenesis progressed through meiosis to the round spermatid stage. Spermatids were selected based on morphological criteria and microinjected into mature, activated bovine oocytes. Although a high percentage of parthenogenesis was observed, approximately 50 percent of embryos were diploid based on karyotype and approximately 35 percent of embryos were positive for the SRY gene, demonstrating contribution of the injected presumptive spermatid. However, morphological assessment of cultured testis cells revealed very few definitive spermatogonia or spermatocytes, indicating the in vitro culture
conditions were inefficient in maintaining germ cell viability and/or proliferation. We are currently looking at a variety of culture conditions in an effort to improve the efficiency of our in vitro spermatogenesis system. These conditions include initial tissue handling and transport(on ice versus 37 degrees C), dissociation procedure (mechanical versus enzymatic), incubation temperature (32 versus 37 degrees C), alginate encapsulation, and addition of pro-survival and anti-apoptotic factors. Results are very preliminary, but seem to suggest that nearly 70 percent of germ cells are apoptotic within 2.5 hr after castration. Germ cell survival during the first week of culture appears to be improved when the testis is mechanically reduced to approximately 1 cubic mm pieces rather than enzymatically dissociated. In addition to evaluating culture conditions, density gradient separation using a STAPUT apparatus is being used to isolate and characterize germ cells from the pubertal testis,
including the use of immunocytochemistry to identify specific germ cell types.
Impacts
A completely in vitro culture system for spermatogenesis will provide a basis for mechanistic studies of spermatogenesis, a model for studying and manipulating spermatogenesis in vitro and in vivo, the potential for genetic modification of the male germ line, and an approach to treating selected causes of male-factor infertility.
Publications
- Parks J.E., Lee D.R., Huang, S., and Kaproth, M.T. 2003. Prospects for spermatogenesis in vitro. Theriogenology 59:73-86.
- Bartholomew, R.A. 2003. Germ cell markers in cattle, Bos taurus:A study of the stem and pluripotent cell-like markers CD34, c-kit, Oct-4, and Vasa in fetal bovine germ cells from 25-120 days of gestation. Ph.D. Dissertation, Cornell University, Ithaca, NY.
- Lee, D.R., Parks, J.E., Lim, J.J., Yoon, H., Ko, J.J., Kim, K.S. 2003. Regulation of the proliferation and differentiation of mouse spermatogonial stem cells by LIF/bFGF and FSH during in vitro culture. Annual Meeting for the American Society for Reproductive Medicine, San Antonio, TX (Abstr.).
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Progress 01/01/02 to 12/31/02
Outputs
We demonstrated the expression of spermatid-specific genes (protamine and transition protein 1) by alginate-encapsulate neonatal bull testis cells after 10 weeks in culture, suggesting that meiosis had occurred. While identifiable germ cells in these cultures were very sparse, some indication of acrosome development was observed based on morphology (diameter and nucleus/cytoplasm ratio), PAS staining of the acrosomic granule, and PNA staining. Following round spermatid injection (ROSI) with presumptive spermatids produced in vitro, 50 percent of blastocysts produced were diploid and 37 percent were Y-chromosome positive demonstrating the contribution of haploid spermatids in a high percentage of embryos. Efforts to improve the efficiency of the culture system have included the replacement of bovine testis serum in the culture medium and numerous combinations of FSH and testosterone relative to the initiation of culture. These modifications have not been effective to
date. Efforts to identify molecular markers for spermatogonial stem cells based on known markers for hematopoitic stem cells and other germ cell markers are ongoing. The germ cell-specific marker VASA has been identified in developing bovine fetal gonads and we successfully cloned the bovine gene for the first time.
Impacts
In vitro conditions which support male germ cell survival and progression through spermatogonial proliferation and meiosis provide a basis for genetically modifying the male germ line in the bull.
Publications
- Parks, John E., Lee, Dong Ryul, Kaproth, M.K., and Huang, Stephanie. 2003. Prospects for spermatogenesis in vitro. Theriogenology 59:73-86.
- Bartholomew, R.A. and Parks, John E. 2002. The cloning of bovine VASA in fetal gonads. Biol. Reprod. 66(Suppl.1):161.
- Kaproth, M., Lee, D.R., and Parks, John E. 2002. Transplantation of testicular explants from prepubertal bulls to nude mice and ex situ production of haploid germ cells over a 20-week period. ASAS-ADSA Joint Ann Mtg 2002; p. 302 (abstr.).
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Progress 01/01/01 to 12/31/01
Outputs
The long-term objective of this research is to develop an in vitro tissue culture system to study and exploit the mechanistic details of mammalian spermatogenesis. Major objectives are to characterize the histological and cytological progression and organization of bovine spermatogenic cells produced in vitro; optimize culture conditions to complete all phases of spermatogenesis (spermatocytogenesis, meiosis, and spermiogenesis) in vitro; determine the developmental competence in vitro and in vivo of bovine embryos produced with in vitro-derived spermatids; and initiate procedures for stable transfection and gene targeting of bovine germ cells in culture. Initial experiments demonstrated dissociation of testis from neonatal bulls followed by agglutination and alginate encapsulation resulted in aggregates of Sertoli and germ cells which remained intact for up to 14 weeks in culture. Between 5 and 10 weeks of culture, expression of haploid specific genes protamine 2 and
transition protein-1 was detected in cultures initiated with either fresh or cryopreserved testis cells. Presence of presumptive spermatids was suggested based on ploidy analysis using flow cytometry but histological preparations indicated that germ cells were present in very low numbers.
Impacts
These studies will provide an in vitro system for studying the mechanistic details of spermatogenesis and provide a culture system which will permit genetic modification of the male germ line.
Publications
- Lee, D. R., Kaproth, M., Parks, John E. 2000. In vitro production of haploid germ cells from testicular cells of neonatal bulls (abstract). Biol. Reprod. 62 (Suppl. 1):594.
- D. R., Kaproth, M. T., and Parks, John E. 2001. In vitro production of haploid germ cells from fresh or frozen-thawed testicular cells of neonatal bulls. Biol. Reprod. (65:834-839).
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