Progress 07/01/10 to 05/31/13
Outputs
Target Audience: Cattle producers, Scientists, extension agents, educators Changes/Problems: Project is being terminated upon leaving Washington State University in June 2013 What opportunities for training and professional development has the project provided? Three graduate students and five undergraduate students were trained during the time period of this project. How have the results been disseminated to communities of interest? Data from the project was published in peer-reviewed journals. Data from the project was presented at international scientific meetings including Society for the Study of Reproduction. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Through the investigation of the effect of VEGF on testis development and spermatogenesis wehave improved our understanding of factors that are critical for maximal sperm production and testis size in bulls. We have determined the temporal expression pattern of VEGF, its receptors (VEGFR1 and VEGFR2) and its cofactors neuropilin1 and neuropilin2. We have functional data to demonstrate that blocking the normal expression pattern of VEGF disrupts spermatogonial stem cell activity. We know that VEGF is an important factor for the development and maintenance of sperm production in males.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Caires KC, de Avila JM, McLean DJ. 2013. Functional analysis of spermatogonial stem cells in the seminiferous tubule of the adult mouse. BioResearch Open Access, 1(5):222-230.
Murphey P, McLean DJ, McMahan CA, Walter CA, McCarrey JR. 2013. Enhanced genetic integrity in mouse germ cells. Biol Reprod 88(1):1-8.
Doyle TJ, Bowman JL, Windell VL, McLean DJ, Kim KH. 2013. Transgenerational Effects of Di-(2-ethylhexyl) Phthalate on testicular germ cell associations and spermatogonial stem cells in mice. Biol Reprod, 88(5):112.
Fuerst, EP, Morris CF, Dasgupta J, McLean DJ. Optimizing experimental design using the house mouse (Mus musculus) as a model for determining grain feeding preferences. J. Food Sci, in press.
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Progress 01/01/12 to 12/31/12
Outputs
OUTPUTS: Vascular endothelial growth factor-A (VEGF) is a hypoxia-inducible peptide essential for angiogenesis, and targets nonvascular cells in a variety of tissues and cell types. The objective of the current study was to determine the function of VEGF in regulating the establishment of spermatogenesis. We hypothesized that VEGF isoforms are important for the early formation and differentiation of spermatogonial stem cells in the testis. The first step in the formation of the spermatogonial stem cell population is the conversion of fetal germ cells called gonocytes into spermatogonial stem cells. This process is essential for male fertility and occurs during the first five days after birth in mice and during the first two to three months of age in cattle. The locally produced signals for the cell differentiation process are not known. VEGFA is expressed in the seminiferous tubules of bull calves at eight to 12 weeks after birth but it is not expressed in the interstitial cells at this time. Somatic Sertoli cells and germ cells are positive for VEGFA eight weeks after birth and this expression continues for several more weeks until 12 weeks of life when no VEGFA can be detected in the germ cells of the testis while Sertoli cells continue to express the protein.<p> The objective of the present study was to investigate vascular endothelial growth factor A (VEGFA) isoform regulation of cell fate decisions of spermatogonial stem cells (SSC) in vivo. The expression pattern and cell-specific distribution of VEGF isoforms, receptors, and coreceptors during testis development postnatal d 1-180 suggest a nonvascular function for VEGF regulation of early germ cell homeostasis. Populations of undifferentiated spermatogonia present shortly after birth were positive for VEGF receptor activation as demonstrated by immunohistochemical analysis. Thus, we hypothesized that proangiogenic isoforms of VEGF (VEGFA(164)) stimulate SSC self-renewal, whereas antiangiogenic isoforms of VEGF (VEGFA(165)b) induce differentiation of SSC. To test this hypothesis, we used transplantation to assay the stem cell activity of SSC obtained from neonatal mice treated daily from postnatal d 3-5 with: 1) vehicle, 2) VEGFA(164), 3) VEGFA(165)b, 4) IgG control, 5) anti-VEGFA(164), and 6) anti-VEGFA(165)b. SSC transplantation analysis demonstrated that VEGFA(164) supports self-renewal, whereas VEGFA(165)b stimulates differentiation of mouse SSC in vivo. Gene expression analysis of SSC-associated factors and morphometric analysis of germ cell populations confirmed the effects of treatment on modulating the biological activity of SSC. These findings indicate a nonvascular role for VEGF in testis development and suggest that a delicate balance between VEGFA(164) and VEGFA(165)b isoforms orchestrates the cell fate decisions of SSC. Future in vivo and in vitro experimentation will focus on elucidating the mechanisms by which VEGFA isoforms regulate SSC homeostasis. PARTICIPANTS: D.J. McLean (Project Leader),<br> D.M.deAvila, J. deAvila, K.C. Caires (Berry College),<br> Andrea Cupp (U. of Nebraska),<br> K. Yenick TARGET AUDIENCES: Cattle producers, Scientists, extension agents, educators PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
A. Through the investigation of the effect of VEGF on testis development and spermatogenesis we will improve our understanding of factors that are critical for maximal sperm production and testis size in bulls.<p> B. Through the investigation of factors that regulate Sertoli cell proliferation and differentiation we will increase the number of sperm produced by males throughout their lifetime.<p> C. Through the identification of factors that regulate the spermatogonial stem cell niche in the testis and stem cell activity, we will be able to increase the number of sperm produced by males throughout their lifetime.
Publications
- Oki A., D.J.McLean. 2012. Exploiting multimedia in reproductive science education: research findings. Reproduction in Domestic Animals 47(Suppl 4):38-45.
- Caires K.C., J.M.Deavila, D.J.McLean. 2012. VEGFA family isoforms regulate spermatogonial stem cell homeostasis in vivo. Endocrinology. 153(2):887-900.
- McCarrey J., D.J.McLean 2012. Enhanced genetic integrity in mouse germ cells. Biology of Reproduction. 88(1):1-8.
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Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: Vascular endothelial growth factor-A (VEGF) is a hypoxia-inducible peptide essential for angiogenesis, and targets nonvascular cells in a variety of tissues and cell types. The objective of the present study was to investigate VEGFA isoform regulation of cell fate decisions of spermatogonial stem cells (SSC) in vivo. The expression pattern and cell specific distribution of VEGF isoforms, receptors and co-receptors during testis development (P1-P180) suggest a non-vascular function for VEGF regulation of early germ cell homeostasis. Populations of undifferentiated spermatogonia present shortly after birth were positive for VEGF receptor activation as demonstrated by immunohistochemical analysis. Thus, we hypothesized that pro-angiogenic isoforms of VEGF (VEGFA164) stimulate SSC self-renewal, whereas anti-angiogenic isoforms of VEGF (VEGFA165B) induce differentiation of SSCs. To test this hypothesis, we used transplantation to assay the stem cell activity of SSCs obtained from neonatal mice treated daily from P3-P5 with: 1) vehicle, 2) VEGFA164, 3) VEGFA165B,4) IgG control, 5) anti-VEGFA164 and 6) anti-VEGFA165B. SSC transplantation analysis demonstrated that VEGFA164 supports self-renewal, whereas VEGFA165B stimulates differentiation of mouse SSCs in vivo. Gene expression analysis of SSC associated factors and morphometric analysis of germ cell populations confirmed the effects of treatment on modulating the biological activity of SSCs. These findings indicate a non-vascular role for VEGF in testis development and suggest that a delicate balance between VEGFA164 and VEGFA165B isoforms orchestrates the cell fate decisions of SSCs. Future in vivo and in vitro experimentation will focus on elucidating the mechanisms by which VEGFA isoforms regulate SSC homeostasis. PARTICIPANTS: D.J. McLean (Project Leader), J. deAvila, D. de Avila, K. Yenick, K.C. Caires, J.Broady TARGET AUDIENCES: Cattle producers, Scientists, extension agents, educators PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
A. Through the investigation of the effect of VEGF on testis development and spermatogenesis we will improve our understanding of factors that are critical for maximal sperm production and testis size in bulls. B. Through the investigation of factors that regulate Sertoli cell proliferation and differentiation we will increase the number of sperm produced by males throughout their lifetime. C. Through the identification of factors that regulate the spermatogonial stem cell niche in the testis and stem cell activity, we will be able to increase the number of sperm produced by males throughout their lifetime.
Publications
- Caires, K.C., A.Cupp, J.M.Deavila, and D.J.McLean. 2011. VEGFA family isoforms regulate spermatogonial stem cell homeostasis in vivo. Endocrinology. . Caires, K.C., J.M.Deavila, C.Shima, and D.J.McLean. 2011. Acute ethanol exposure affects spermatogonial stem cell homeostasis in neonatal mice. Reproductive Toxicology.
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: Vascular endothelial growth factor-A (VEGF) is a hypoxia-inducible peptide essential for angiogenesis, and targets nonvascular cells in a variety of tissues and cell types. The objective of the present study was to investigate VEGFA isoform regulation of cell fate decisions of spermatogonial stem cells (SSC) in vivo. The expression pattern and cell specific distribution of VEGF isoforms, receptors and co-receptors during testis development (P1-P180) suggest a non-vascular function for VEGF regulation of early germ cell homeostasis. Populations of undifferentiated spermatogonia present shortly after birth were positive for VEGF receptor activation as demonstrated by immunohistochemical analysis. Thus, we hypothesized that pro-angiogenic isoforms of VEGF (VEGFA164) stimulate SSC self-renewal, whereas anti-angiogenic isoforms of VEGF (VEGFA165B) induce differentiation of SSCs. To test this hypothesis, we used transplantation to assay the stem cell activity of SSCs obtained from neonatal animals treated daily from P3-P5 with: 1) vehicle, 2) VEGFA164, 3) VEGFA165B,4) IgG control, 5) anti-VEGFA164 and 6) anti-VEGFA165B. SSC transplantation analysis demonstrated that VEGFA164 supports self-renewal, whereas VEGFA165B stimulates differentiation of mouse SSCs in vivo. Gene expression analysis of SSC associated factors and morphometric analysis of germ cell populations confirmed the effects of treatment on modulating the biological activity of SSCs. These findings indicate a non-vascular role for VEGF in testis development and suggest that a delicate balance between VEGFA164 and VEGFA165B isoforms orchestrates the cell fate decisions of SSCs. Future in vivo and in vitro experimentation will focus on elucidating the mechanisms by which VEGFA isoforms regulate SSC homeostasis. PARTICIPANTS: Participants: D.J. McLean (Project Leader), D.M.deAvila, J. deAvila, N. Cummings, K.C. Caires, J.Broady TARGET AUDIENCES: Cattle producers, scientists, extension agents, educators PROJECT MODIFICATIONS: Not Reported
Impacts
A. Through the investigation of the effect of VEGF on testis development and spermatogenesis we will improve our understanding of factors that are critical for maximal sperm production and testis size in bulls. B. Through the investigation of factors that regulate Sertoli cell proliferation and differentiation we will increase the number of sperm produced by males throughout their lifetime. C. Through the identification of factors that regulate the spermatogonial stem cell niche in the testis and stem cell activity, we will be able to increase the number of sperm produced by males throughout their lifetime.
Publications
- Caires, K.C., J.W.Broady, and D.J.McLean. 2010. Maintaining the male germline: regulation of spermatogonial stem cells. The Journal of Endocrinology. 205(2):133-145.
- Michelizzi, V.N., M.V.Dodson, Z.Pan, M.E.Amaral, J.J.Michal, D.J.McLean, J.E.Womack, and Z.Jiang. 2010. Water buffalo genome science comes of age. International Journal of Biological Sciences 6:333-349.
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: Vascular endothelial growth factor-A (VEGF) is a hypoxia-inducible peptide essential for angiogenesis, and targets nonvascular cells in a variety of tissues and cell types. The objective of the current study was to determine the function of VEGF in regulating the establishment of spermatogenesis. We hypothesized that VEGF isoforms are important for the early formation and differentiation of spermatogonial stem cells in the testis. The first step in the formation of the spermatogonial stem cell population is the conversion of fetal germ cells called gonocytes into spermatogonial stem cells. This process is essential for male fertility and occurs during the first five days after birth in mice and during the first two to three months of age in cattle. The locally produced signals for the cell differentiation process are not known. VEGFA is expressed in the seminiferous tubules of bull calves at eight to 12 weeks after birth but it is not expressed in the interstitial cells at this time. Somatic Sertoli cells and germ cells are positive for VEGFA eight weeks after birth and this expression continues for several more weeks until 12 weeks of life when no VEGFA can be detected in the germ cells of the testis while Sertoli cells continue to express the protein. Interestingly, at 24 weeks of life in calves, when germ cell meiosis is active in the testis, VEGFA protein is present in meiotic germ cells but not the less differentiated spermatogonia. This pattern of expression, present in meiotic germ cells, Sertoli cells and Leydig cells with no expression in spermatogonia continues throughout life. The expression of VEGFA in germ cells during the time period when spermatogonial stem cells differentiate from gonocytes (four to eights weeks after birth) supports our hypothesis that VEGFA is an important autocrine factor for the formation of the spermatogonial stem cell population. The VEGF isoform receptors Flt1 and KDR are expressed in both the germ and somatic cells of the bull testis during development from six weeks after birth until animals are adults. Germ cells demonstrate a strong expression of both FLT1 and KDR, supporting our hypothesis that VEGF isoform signaling occurs in these cells to regulate germ cell function during the formation of the spermatogonial stem cell population. The cofactors NRP1 and NRP2 are expressed in the bull testis during testis development. NRP1 is expressed in germ and somatic cells in the testis at four weeks after birth but no expression is detected in the testis at eight weeks. Interestingly, the expression of NRP1 returns at ten weeks after birth in both germ and somatic cells of the testis. Expression of NRP1 continues in spermatogonia until 24 weeks after birth and then decreases to undetectable levels in the testis of sexually mature animals. The unique expression pattern of NRP1 during the first weeks after birth suggests this protein is involved in early germ cell differentiation, potentially the formation of spermatogonial stem cells. NRP2 is expressed in the testis in both germ cells and somatic cells from birth throughout development and in adult animals. PARTICIPANTS: D.J. McLean (Project Leader), J. deAvila, L-Y. Chen, K.C. Caires, M. Bellefeuille, J.Broady TARGET AUDIENCES: Cattle producers, scientists, extension agents, educators PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
A. Through the investigation of the effect of VEGF on testis development and spermatogenesis we will improve our understanding of factors that are critical for maximal sperm production and testis size in bulls. B. Through the investigation of proteins expressed in accessory sex glands in cattle that influence bull fertility, we will improve our understanding of sperm-protein interactions that regulate sperm function in the female reproductive tract. C. Through the investigation of factors that regulate Sertoli cell proliferation and differentiation we will increase the number of sperm produced by males throughout their lifetime. D. Through the identification of factors that regulate the spermatogonial stem cell niche in the testis and stem cell activity, we will be able to increase the number of sperm produced by males throughout their lifetime.
Publications
- Caires, K.C., J.Oatley, and D.J.McLean. 2009. Advances in the investigation of cellular, molecular and genomic mechanisms regulating testis development and spermatogenesis in livestock. Reproductive Genomics of Domestic Animals. Ed. Zhihua Jiang.
- Caires, K.C., J.M.Deavila, and D.J.McLean. 2009. Vascular endothelial growth factor A (VEGF) regulates germ cell survival during the establishment of spermatogenesis in the bovine testis. Reproduction : the Official Journal of the Society for the Study of Fertility. 138(4):667-677.
- Caires KC, de Avila JM, McLean DJ. Vascular endothelial growth factor A (VEGF) regulates germ cell survival during the establishment of spermatogenesis in the bovine testis. Reproduction. 2009 138(4):667-77.
- McLean D, Caires K, Broady J. 2010. Maintaining the male germline: regulation of spermatogonial stem cells. J Endocrinol. Feb 10. [Epub ahead of print].
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: Vascular endothelial growth factor-A (VEGF) is a hypoxia-inducible peptide essential for angiogenesis. It targets nonvascular cells in a variety of tissues and cell types. The objective of the current study was to determine the function of VEGF in regulating the establishment of spermatogenesis. Using an explant tissue culture and grafting approach to evaluate VEGF treatment addressed the hypothesis that VEGF could regulate germ cell function. We demonstrated that VEGF and its receptors are expressed in germ and somatic cells in the bovine testis. Regardless of treatment, testis tissue graft growth and androgen biosynthesis was not different and all grafts contained spermatogonia and meiotic germ cells. In contrast to controls, the extent of germ cell differentiation was 7-fold greater in bovine testis tissue treated with VEGF, and these grafts contained more meiotic germ cells and spermatids. VEGF treatment resulted in 28% more germ cell survival, as evidenced by fewer numbers of seminiferous tubules containing only Sertoli cells, when compared to controls. Inhibiting the biological activity of VEGF in vitro confirmed the role of VEGF in promoting germ cell survival and VEGF treatment. The BCL2 family of proteins functions as regulators of cell survival and is comprised of both anti-apoptotic (BCL2, BCLXL) and pro-apoptotic (BAX, Bad) members. Therefore, the ratio of mRNA expression between the pro-survival BCL2 and pro-apoptotic BAX was measured in control and VEGF treated bovine testis tissues to determine if VEGF regulates germ cell survival in the bovine testis. When compared to controls, treatment with VEGF significantly increased the ratio of BCL2:BAX expression at 3, 6 and 24 hrs. VEGF can protect extravascular cells from hypoxia-induced cell death; therefore we investigated the mRNA expression of pro-apoptotic Bcl-2/adenovirus E1B 19 kDa interacting protein (BNIP3), a hypoxia responsive gene, in response to VEGF treatment. Regardless of treatment, BNIP3 mRNA was significantly higher (p<0.05) at all time points during explant culture in comparison to the 0-hr time point. When compared to controls, treatment with VEGF significantly decreased BNIP3 mRNA transcript levels at 3, 6 and 24 hrs, respectively. Thus, VEGF treatment promoted two independent means for cell survival in bovine testis tissue. KDR and Flt-1 proteins are localized to germ and somatic cells in the bovine testis, thus we assayed whether VEGF treatment would affect the expression of these genes during explant culture. In comparison to baseline expression (0-hr), the expression of KDR and Flt-1 were significantly induced (p<0.05) at 3 hrs, regardless of treatment. However, VEGF treatment did significantly increase the expression of Flt-1 and KDR mRNA at 24 hrs when compared to controls. These data support the conclusion that VEGF is important in germ cell survival in the developing bovine testis. PARTICIPANTS: D.J. McLean (Project Leader), J. deAvila, L-Y. Chen, K.C. Caires, M. Bellefeuille, J. Broady TARGET AUDIENCES: Cattle producers, Scientists, Extension agents, Educators PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
A. Through the investigation of the effect of VEGF on testis development and spermatogenesis, we will improve our understanding of factors that are critical for maximal sperm production and testis size in bulls. B. Through the investigation of proteins expressed in accessory sex glands in cattle that influence bull fertility, we will improve our understanding of sperm-protein interactions that regulate sperm function in the female reproductive tract. C. Through the investigation of factors that regulate Sertoli cell proliferation and differentiation, we will increase the number of sperm produced by males throughout their lifetime. D. Through the identification of factors that regulate the spermatogonial stem cell niche in the testis and stem cell activity, we will be able to increase the number of sperm produced by males throughout their lifetime.
Publications
- McLean, D.J. 2008. Spermatogonial stem cell transplantation and testicular function. Methods in Molecular Biology: Germline stem cell protocols. Ed. Steven Hu. Humana Press. 149-162.
- McLean, D.J. 2008. Endocrine regulation of the establishment of spermatogenesis in pigs. Reproduction in Domestic Animals 43(2):280-287.
- McLean, D.J. 2008. Cell Biology Symposium: The Role of MicroRNA in Cell Function. Journal of Animal Science.
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Progress 01/01/07 to 12/31/07
Outputs
Recent work in our lab has suggested non-endothelial cell targets for VEGF-164 in testicular germ cells. We treated bovine testis tissue grafted onto the backs of mice with rmVEGF-164 to determine if it would stimulate blood vessel growth and eventual germ cell differentiation in the grafted testis tissue. Treatment with VEGF resulted in more germ cell differentiation as indicated by the number of elongate spermatids in grafted testis tissue but there was no difference in the number of blood vessels in the grafted tissue. Therefore, we hypothesized that VEGF stimulates germ cell differentiation in pre-pubertal bovine testes. To test this hypothesis, the objective of this study was to determine the effect of VEGF on germ cell differentiation in the bovine testis. To accomplish this objective, we used the experimental approach of treating bovine testis tissue in culture with VEGF prior to grafting to determine if VEGF actions on germ cell differentiation were independent
of blood vessel growth into the grafted tissue. We hypothesized that treating bovine testis tissue with VEGF in explant cultures prior to grafting would result in more differentiating germ cells in the testis tissue. Testicular parenchyma obtained from 4- and 8-wk bovine donors were placed in explant culture on floating filters for 5-days prior to grafting, and treated every two days with recombinant mouse VEGF-164 (rmVEGF; 0, 100, 200 ng/ml) with media change. Following culture and treatment, testis tissues were grafted onto the backs of immunodeficient mice. Recipient mice were sacrificed when testis xenografts reached a tissue age of 32 weeks. Following sacrifice, grafts were weighed and evaluated for the extent of germ cell differentiation and mouse vesicular gland weights were recorded as a bioassay for androgen synthesis. No differences existed between donor age and grafting period so these datasets were pooled. Seminiferous tubules of testis grafts treated with VEGF contained
more differentiating germ cells as evident by a greater percentage of meiotic germ cells and spermatids when compared to controls (p < 0.05). However, no differences existed in the growth and androgen biosynthesis by testis grafts. These data provide direct evidence that VEGF has a functional role in promoting germ cell survival and differentiation in bovine testis xenografts. The underlying mechanisms responsible for the VEGF-induced germ cell differentiation are the focus of future experiments.
Impacts
Reproductive efficiency is important when evaluating the economic impact of the production of milk or beef. Improved ability to generate animals with desirable genetic traits would help in reducing the number of animals needed to produce desirable outcomes. This research will help answer questions about how sperm are produced by bulls and develop novel ways to evaluate sperm production. Information gained will aid in the genetic improvement of cattle and accelerate the process of using biotechnology in beneficial ways for cattle production. Results will be shared with potential beneficiaries including veterinarians, cattle producers and cattle technology companies by the use of collaborative projects, presentations at national meetings and extension efforts throughout the state.
Publications
- Schmidt, J.A., de Avila, J., and McLean, D.J. 2007. Analysis of gene expression in bovine testis tissue prior to ectopic testis tissue xenografting and during the grafting period. Biology of Reproduction 76:1071-1080.
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Progress 01/01/06 to 12/31/06
Outputs
Testis development requires a coordinated sequence of events resulting in germ-somatic cell interactions that support proper interactions differentiation. The long-term objective of the research program is to develop new approaches and investigate the molecular and biological regulation of germ and somatic cells involved in mammalian spermatogenesis. The research objectives of the project include continued investigation of factors regulating bovine and porcine spermatogenesis with the use of ectopic grafting of testicular tissue. Ectopic grafting of bovine tissue is a technique in which small pieces of testis tissue is surgically placed under the skin on the backs of immunodeficient (nude) mice. Ectopic subcutaneous grafts of bovine and porcine testis tissue survive, develop blood vessel growth from host tissue, increase in size and produce testosterone on the backs of castrated, nude mice. The goals of the research project this year were 1. extending the grafting
period to increase sperm production in grafts, 2. investigation of the tissue age/grafting period, 3. determine if altering blood vessel growth into the tissue affects spermatogenesis and 4. determine if increasing the number of spermatogonial stem cells in tissue prior to grafting increases spermatogenesis. A significant increase in spermatogenesis in grafts was observed following treatment of tissue with factors involved in blood vessel growth. However, blood vessel growth was only marginally improved by this treatment. These results suggest that vascular growth factors influence the differentiation of somatic and germ cells in the testis and could be critical for efficient sperm production in adults. Extending the grafting period from 24 to 36 weeks resulted in an increase in the number of seminiferous tubules with elongating spermatids from 2% to 10% in two-week donor tissue. These data demonstrate that both donor age and grafting period are important factors regulating the
maturation of bovine testis xenografts, indicating that intrinsic differences exist within these donor ages. In conclusion, these studies have: 1. demonstrated vascular growth factors regulate germ cell differentiation in the testis; 2. demonstrated that important developmental changes occur from the 2-8 week age in the bovine testis; 3. demonstrated that in vitro culture of testis tissue prior to grafting does not negatively affect sperm production in testis tissue grafts, and 4. demonstrated that cryopreserved porcine testis tissue produces sperm after grafting. Collectively, these results provide us with new avenues to pursue such as exploring the mechanisms that regulate early testicular differentiation in bovine testes and how vascular growth factors regulate somatic and germ cell differentiation in the bovine testis. Grafting of pig testis tissue demonstrated that testis tissue from pigs at day 14 after birth has a high potential for germ cell production compared to tissue from
other ages. Current studies are investigating if Sertoli cell differentiation regulates germ cell production in pig grafts.
Impacts
Reproductive efficiency is important when evaluating the economic impact of the production of milk or beef. Improved ability to generate animals with desirable genetic traits would help in reducing the number of animals needed to produce desirable outcomes. This research will help answer questions about how sperm are produced by bulls and develop novel ways to evaluate sperm production. Information gained will aid in the genetic improvement of cattle and accelerate the process of using biotechnology in beneficial ways for cattle production. Results will be shared with potential beneficiaries including veterinarians, cattle producers and cattle technology companies by the use of collaborative projects, presentations at national meetings and extension efforts throughout the state.
Publications
- Schmidt J.A., J.M. de Avila and D.J. McLean. 2006. Effect of vascular endothelial growth factor and testis tissue culture on spermatogenesis in bovine ectopic testis xenografts. Biol. Reprod. 75(2):167-175.
- Schmidt J.A., J.M. de Avila and D.J. McLean. 2006. Grafting period and donor age affect the potential for spermatogenesis in bovine ectopic testis xenografts. Biol. Reprod. 75(2):160-166.
- McLean D.J. 2006. Vertebrate reproductive stem cells: Recent insights and technological advances. Semin. Cell Dev. Biol. 17(4):534-539.
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Progress 01/01/05 to 12/31/05
Outputs
The long-term objective of the research program is to develop new approaches and investigate the molecular and biological regulation of germ and somatic cells involved in mammalian spermatogenesis. The research objectives of the project include continued investigation of factors regulating bovine and porcine spermatogenesis with the use of ectopic grafting of testicular tissue. Ectopic grafting of bovine tissue is a technique in which small pieces of bull testis tissue is surgically placed under the skin on the backs of immunodeficient (nude) mice. Ectopic subcutaneous grafts of bovine and porcine testis tissue survive, develop blood vessel growth from host tissue, increase in size and produce testosterone on the backs of castrated, nude mice. The goals of the research project this year were 1. extending the grafting period to increase sperm production in grafts, 2. investigation of the tissue age/grafting period, 3. determine if altering blood vessel growth into the
tissue affects spermatogenesis and 4. determine if increasing the number of spermatogonial stem cells in tissue prior to grafting increases spermatogenesis. A significant increase in spermatogenesis in grafts was observed following treatment of tissue with factors involved in blood vessel growth. However, blood vessel growth was only marginally improved by this treatment. These results suggest that vascular growth factors influence the differentiation of somatic and germ cells in the testis and could be critical for efficient sperm production in adults. Extending the grafting period from 24 to 36 weeks resulted in an increase in the number of seminiferous tubules with elongating spermatids from 2% to 10% in two-week donor tissue. These data demonstrate that both donor age and grafting period are important factors regulating the maturation of bovine testis xenografts, indicating that intrinsic differences exist within these donor ages. In conclusion, these studies have: 1. demonstrated
vascular growth factors regulate germ cell differentiation in the testis; 2. demonstrated that important developmental changes occur from the 2-8 week age in the bovine testis; 3. demonstrated that in vitro culture of testis tissue prior to grafting does not negatively effect sperm production in testis tissue grafts, and 4 . demonstrated that cryopreserved porcine testis tissue produces sperm after grafting. Collectively, these results provide us with new avenues to pursue such as exploring the mechanisms that regulate early testicular differentiation in bovine testes and how vascular growth factors regulate somatic and germ cell differentiation in the bovine testis.
Impacts
Knowledge gained from this research will enhance our understanding of spermatogenesis in livestock species to improve reproductive efficiency in these animals. We can use these techniques to determine the factors that influence efficient sperm production and develop alternative reproductive technologies for the production of genetically modified livestock. Genetically modified (transgenic) livestock will provide novel ways to produce therapeutic products for animal or human health in large quantities. Animals capable of producing useful products would be very valuable. In addition, the use of bull testis tissue provides a useful model to determine if ectopic testis tissue grafting is suitable to use as an assisted reproductive technology for humans. This technique would be very useful to individuals who have lost sperm production due to cancer treatments or detrimental environmental exposure to toxins. Lastly, we can use this technique to explore ways to overcome
blocks in sperm production that result in male factor infertility and investigate the mechanisms regulating sperm production in mammals.
Publications
- Oatley, J.M., J.J. Reeves, and D.J. McLean. 2005. Establishment of spermatogenesis in neonatal bovine testicular tissue following ectopic xenografting varies with donor age. Biol Reprod. 72:358-364.
- McLean, D.J. 2005. Spermatogonial stem cell transplantation and testicular function. Cell Tissue Res. 322(1):21-31.
- Oatley J.M., D.M. de Avila, A. Tibary, J. Wheaton, D.J. McLean, and J.J. Reeves. 2005. Changes in spermatogenesis and endocrine function in the ram testis due to irradiation and active immunization against LHRH. J. Anim. Sci. 83:604-612.
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Progress 01/01/04 to 12/31/04
Outputs
The long-term objective of the research program is to develop new approaches to investigate the molecular and biological regulation of spermatogonial stem cells (SSCs) and mammalian spermatogenesis. The research objectives of the project this year were to develop ectopic grafting of bovine testicular tissue to determine the optimal conditions to maintain testicular tissue on the backs of the recipient mice for the production of bull spermatozoa and to culture bovine spermatogonial stem cells to investigate the cellular mechanisms that regulate their proliferation and self-renewal. Ectopic grafting of bovine tissue is a technique in which small pieces of bull testis tissue is surgically placed under the skin on the backs of immunodeficient (nude) mice. Ectopic subcutaneous grafts of bovine testis tissue survive, develop blood vessel growth from host tissue, increase in size and produce testosterone on the backs of castrated, nude mice. Ectopic subcutaneous grafts of
bovine testis tissue from 4- and 8-week-old bull calves produce elongate spermatids 24 weeks after grafting. Although testis tissue from 2-week-old bull calf testis tissue had the greatest growth potential, testis tissue from 8-week-old bull calves had the highest percentage of seminiferous tubules with elongate spermatids. In addition, following electroporation with a vector containing the B-galactosidase gene, cells of the bovine seminiferous tubules in grafts express B-galactosidase. Short-term culture of bovine testis tissue using an explant culture system results in an increase of spermatogonial stem cells in the testis tissue as determined following spermatogonial stem cell transplantation. Bovine spermatogonial stem cells were also cultured on a bovine embryonic fibroblast feeder cell line as dispersed cells. Glial cell line derived neurotrophic factor (GDNF) was shown to be an important factor in the regulation of the survival and proliferation of bovine spermatogonial stem
cells. In conclusion, these studies have: 1. confirmed that ectopic grafting of bovine testis tissue on castrated nude mice results in the production of testosterone by grafted tissue and elongate spermatids; 2. shown that genetic manipulation of germ cells by electroporation prior to grafting results in the stable integration and expression of transgenes in germ cells in grafted tissue; 3. shown that testis tissue from 8-week-old bull calves produce more elongate spermatids that testis tissue from 2-, 4-, 12- and 16-week calves, 4. shown that the number of SSCs in bovine testis tissue cultured for 1-2 weeks in an explant culture system survive, express genes important to testis function and significantly increase in number and, 5. shown that GDNF is critical for bovine SSC survival. Collectively, these results provide us with new avenues to pursue, such as exploring the mechanisms that make 8-week-old bull testis tissue better at producing elongate spermatids than other ages after
ectopic testis tissue grafting, what mechanisms regulate SSC survival in explant cultures and if explant culture will improve the ability of bovine testis tissue to produce sperm following grafting.
Impacts
Knowledge gained from this research will enhance our understanding of spermatogenesis in livestock species to improve reproductive efficiency in these animals. We can use these techniques to develop alternative reproductive technologies for the production of genetically modified livestock. Genetically modified (transgenic) livestock will provide novel ways to produce therapeutic products for animal or human health in large quantities. Animals capable of producing useful products would be very valuable. In addition, the use of bull testis tissue provides a useful model to determine if ectopic testis tissue grafting is suitable to use as an assisted reproductive technology for humans. This technique would be very useful to individuals who have lost sperm production due to cancer treatments or detrimental environmental exposure to toxins. Lastly, we can use this technique to explore ways to overcome blocks in sperm production that result in male factor infertility and
investigate the mechanisms regulating sperm production in mammals.
Publications
- Oatley, J.M., D.M. de Avila, J.J. Reeves, and D.J. McLean. Mar 2004. Testis tissue explant culture supports survival and proliferation of bovine spermatogonial stem cells. Biol. Reprod. 70:625-631.
- Oatley, J.M., D.M. de Avila, J.J. Reeves, and D.J. McLean. Aug 2004. Spermatogenesis and germ cell transgene expression in xenografted bovine testicular tissue. Biol. Reprod. 71:494-501.
- Oatley, J.M., J.J. Reeves, and D.J. McLean. Sep 2004. Biological activity of cryopreserved bovine spermatogonial stem cells during in vitro culture. Biol. Reprod. 71:942-947.
- Oatley J.M., J.J. Reeves, and D.J. McLean. 2004. Spermatogenesis and germ cell transgene expression in xenografted bovine testicular tissue. Biol. Reprod. Abst. 70:593.
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Progress 01/01/03 to 12/31/03
Outputs
This project involves grafting testis tissue from immature bulls under the skin on the back of castrated, immunodeficient mice. In addition, the project includes an aim to genetically modify the bovine testis tissue prior to grafting in order to produce genetically modified sperm capable of generating transgenic offspring. We are also investigating the survival and proliferation of bovine spermatogonial stem cells in culture by transplanting these cells into the testes of recipient mice. Bovine testis tissue survives and supports the differentiation of germ cells to produce elongating bovine spermatids 6 months after ectopic grafting on immunodeficient mice. Bovine Leydig cells produce physiological levels of testosterone in recipient mice 3-6 months after grafting. Bovine testis tissue was electroporated 25, 50 and 75 mV with a plasmid containing the beta-galactosidase reporter gene. Beta-galactosidase gene expression was highest in tissue electroporated at 50 mV.
Reporter gene expression was detected in the seminiferous tubules in both somatic Sertoli cells and differentiating germ cells. These data indicate genomic modification of differentiating germ cells prior to grafting may generate transgenic sperm to produce genetically modified offspring. Ectopic grafting of bovine testicular tissue from donors of different ages indicates that the establishment of spermatogenesis in ectopic grafts is consistent with the timing of the establishment of spermatogenesis in the testis of the animal. In addition, testis tissue from 8-week donors had the highest levels of spermatogenesis when compared to 2-, 4-, 12- and 16-week old donors. There was little to no germ cell differentiation in 12 and 16-week old donors. Testis tissue from 2-week old donors had the highest increase in weight but there were very few tubules containing elongate spermatids. Culture of bovine testis tissue explants cultured on floating filters supported the expansion of the
spermatogonial stem cell population after one week of culture. Explants of calf testicular parenchyma were placed on 0.45micron pore membranes in culture and maintained for 1-2wk. Histological examinations of fresh (t0) and cultured tissues revealed morphologically normal seminiferous tubules. Germ cell numbers/tubule increased (P<0.05) during culture when compared to t0, yet germ cell differentiation was not observed. Single cell suspensions were prepared from the testicular tissues at t0 and during culture and transplanted into nude mouse testes to investigate spermatogonial stem cell viability. One-month after transplantation, colonies of round bovine cells were identified in all mouse testes analyzed, indicating survival of spermatogonial stem cells. The average number of resulting colonies in recipient testes was significantly (P<0.05) higher following 1wk of culture compared to t0, and was numerically higher at 2wk of culture compared to t0. This increase in colony numbers over
time in culture indicates spermatogonial stem cell proliferation in vitro. This explant culture system appears to provide an environment that supports survival and proliferation of bovine spermatogonial stem cells.
Impacts
This project may potentially lead to the development of new ways to provide unlimited supply to sperm from sires with desirable traits. In addition, preliminary data indicates sperm in ecotopic testis grafts can be genetically modified. This may provide a novel way to generate transgenic livestock. This technique could provide high numbers of transgenic sperm leading to less expensive approaches to produce genetically modified animals.
Publications
- No publications reported this period
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