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Sequencing of RNA from stallion sperm identifies potential markers of fertility
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Abstracts / Journal of Equine Veterinary Science 43 (2016) S56eS82
19 Sequencing of RNA from stallion sperm identifies potential markers of fertility N.H. Ing 1, K. Konganti 2, N. Ghaffari 3, C.D. Johnson 3, D.W. Forrest 1, C.C. Love 4, D.D. Varner 4 1 Department of Animal Science, Texas A&M University, College Station, Texas, USA; 2 Texas A&M Institute for Genome Sciences and Society, Texas A&M University, College Station, Texas, USA; 3 AgriLife Genomics and Bioinformatics, Texas A&M University, College Station, TX, USA; 4 Large Animal Clinical Sciences, Texas A&M University, College Station, Texas, USA
P. Henney 1, M.H. Troedsson 1, R.F. Cook 1, T. Swerczek 1, E.L. Squires 1, E. Bailey 1, U.B.R. Balasuriya 1 1 Maxwell H. Gluck Equine Research Center, Department of Veterinary Science, University of Kentucky, Lexington, KY, USA; 2 University of Kentucky Veterinary Diagnostic Laboratory, Department of Veterinary Science, University of Kentucky, Lexington, KY, USA; 3 Virus Research and Testing Group, Division of Drug Discovery Research, Korea Research Institute of Chemical Technology, Yuseong-gu, Daejeon, Korea; 4 Department of Veterinary Clinical Medicine, College of Veterinary Medicine, University of Illinois, Urbana, Illinois, USA
Subfertile stallions often require assisted reproductive technologies to improve fertility. One technique to improve fertility is isolation of more dense sperm from less dense sperm by density gradient centrifugation before use in artificial insemination. More dense sperm have superior motility, morphology, chromatin integrity and, compared to unfractionated sperm, they have superior fertilizing ability. Recently, it has been discovered that sperm contain many mRNAs and microRNAs, which are being investigated for development of novel assays to predict fertility. The mRNAs in sperm are fragmented but may reflect the health of the sperm during their development. Sperm also carry microRNAs, which are stably packaged in multiprotein complexes. These are delivered at conception to the zygote and could function as regulators of numerous genes. Studies of human sperm have found that specific RNAs may be correlated with fertility. The current study used sequencing to measure global mRNAs and microRNAs in more dense and less dense sperm from five performance-bred American Quarter Horse stallions of normal fertility and semen parameters. Of the 168 mature microRNAs detected, none were differentially expressed in more dense sperm compared to less dense sperm. For the analysis of the 11,216 mRNAs detected in sperm, we used two bioinformatics methods to measure differential gene expression; one identified 23 mRNAs that had greater concentrations in more dense sperm compared to the less dense sperm (FDR < 0.05). The second identified 163 mRNAs with greater concentrations in more dense sperm and 7 mRNAs with greater concentrations in less dense sperm (FDR < 0.05). Based on these two lists, four gene products that were of moderate to high prevalence were chosen for confirmation of differential expression by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The qRT-PCR analyses determined that SCP2 sterol-binding domain containing 1 (SCP2D1) and spermatogenesis-associated protein 31D1 (SPATA31D1) mRNA concentrations were 6- and 3-fold greater in more dense sperm, respectively, than in less dense sperm (P < 0.02). Conversely, solute carrier family 26 (anion exchanger), member 8 (SLC26A8) mRNA concentrations were 9-fold greater in less dense sperm (P < 0.05). Intriguingly, all three genes are expressed either exclusively or predominantly in sperm. Future studies will explore the functions of the products of these genes in sperm and determine their usefulness as markers of fertility.
Equine arteritis virus (EAV) is the prototype member of the family Arteriviridae, order Nidovirales. Following natural infection, 1070% of the infected stallions can become persistently infected and continue to shed EAV in semen for a variable period of time ranging from several months after infection to life-long. Therefore, carrier stallions play an important role in the maintenance of EAV in equine populations. Even though it has been determined that the EAV carrier state is testosterone-dependent, the mechanism(s) by which EAV establishes persistent infection remains to be elucidated. The objective of this study was to determine the tissue and cellular localization of EAV in the reproductive tract of long-term carrier stallions, as well as the inflammatory response induced during persistent infection. A total of 9 EAV-infected stallions (3 long-term carriers and 6 stallions that stopped viral shedding in semen) were used in this study. EAV sites of persistency were determined by virus isolation, single (EAV nucleocapsid-specific) and dual immunohistochemistry (IHC) performed on various samples collected from the reproductive tract. Furthermore, the inflammatory response was characterized by histopathology and single IHC using a panel of differentiation (CD) markers. Histological lesions were characterized by minimal to mild, focal to multifocal lymphoplasmacytic inflammation. The inflammatory response comprised moderate to high numbers of CD3+ T lymphocytes (with low to moderate, and moderate to high numbers of CD4+ and CD8+ T lymphocytes, respectively), low to moderate numbers of CD25+ T regulatory lymphocytes, minimal to moderate numbers of CD21+ B lymphocytes, and low to moderate numbers of CD83+ dendritic cells. EAV was successfully isolated from the ampulla and other accessory sex glands of carrier stallions including vesicular glands, prostate, and bulbourethral glands; and the ampulla demonstrated to be the tissue with the highest viral titer in the reproductive tract (1.0x103 e 1.7x105 PFU/g). Dual IHC demonstrated the presence of viral antigen in fibroblasts, lymphocytes (mainly CD3+ [CD8+] and CD21+), and other mononuclear cells predominantly in the lamina propria and inflammatory infiltrates in the ampulla. This study unequivocally confirms the ampulla as the primary site of EAV persistence and demonstrates that EAV has a particular tropism for fibroblasts and cells of the mononuclear lineage (including T and B lymphocytes) that participate in the inflammatory response. Interestingly, the glandular epithelium does not seem to play a role in viral maintenance during long-term persistent infection.
Acknowledgments
Key Words: EAV, carrier, tropism
Dr. G. P. Blodgett at the 6666 Ranch and Sheila Teague.
21 Sperm-bound antisperm antibodies affect motility of cooled stallion spermatozoa
Key Words: spermatozoa, RNA, fertility
20 Sites of persistent equine arteritis virus infection in the reproductive tract of the long-term carrier stallion M. Carossino 1, A.T. Loynachan 2, J.R. Campos 1, B. Nam 1, I.F. Canisso 4, Y.Y. Go 1, 3, P.J. Timoney 1, K.M. Shuck 1,
M.S. Ferrer 1, 3, L.M.J. Miller 2, 3, A. George 3, M.E. Wilkerson 3 1 Department of Large Animal Medicine, University of Georgia, Athens, GA, USA; 2 Oklahoma Equine Hospital, Washington, OK, USA; 3 College of Veterinary Medicine, Kansas State University, Manhattan, KS, USA
Abstracts / Journal of Equine Veterinary Science 43 (2016) S56eS82
19 Sequencing of RNA from stallion sperm identifies potential markers of fertility N.H. Ing 1, K. Konganti 2, N. Ghaffari 3, C.D. Johnson 3, D.W. Forrest 1, C.C. Love 4, D.D. Varner 4 1 Department of Animal Science, Texas A&M University, College Station, Texas, USA; 2 Texas A&M Institute for Genome Sciences and Society, Texas A&M University, College Station, Texas, USA; 3 AgriLife Genomics and Bioinformatics, Texas A&M University, College Station, TX, USA; 4 Large Animal Clinical Sciences, Texas A&M University, College Station, Texas, USA
P. Henney 1, M.H. Troedsson 1, R.F. Cook 1, T. Swerczek 1, E.L. Squires 1, E. Bailey 1, U.B.R. Balasuriya 1 1 Maxwell H. Gluck Equine Research Center, Department of Veterinary Science, University of Kentucky, Lexington, KY, USA; 2 University of Kentucky Veterinary Diagnostic Laboratory, Department of Veterinary Science, University of Kentucky, Lexington, KY, USA; 3 Virus Research and Testing Group, Division of Drug Discovery Research, Korea Research Institute of Chemical Technology, Yuseong-gu, Daejeon, Korea; 4 Department of Veterinary Clinical Medicine, College of Veterinary Medicine, University of Illinois, Urbana, Illinois, USA
Subfertile stallions often require assisted reproductive technologies to improve fertility. One technique to improve fertility is isolation of more dense sperm from less dense sperm by density gradient centrifugation before use in artificial insemination. More dense sperm have superior motility, morphology, chromatin integrity and, compared to unfractionated sperm, they have superior fertilizing ability. Recently, it has been discovered that sperm contain many mRNAs and microRNAs, which are being investigated for development of novel assays to predict fertility. The mRNAs in sperm are fragmented but may reflect the health of the sperm during their development. Sperm also carry microRNAs, which are stably packaged in multiprotein complexes. These are delivered at conception to the zygote and could function as regulators of numerous genes. Studies of human sperm have found that specific RNAs may be correlated with fertility. The current study used sequencing to measure global mRNAs and microRNAs in more dense and less dense sperm from five performance-bred American Quarter Horse stallions of normal fertility and semen parameters. Of the 168 mature microRNAs detected, none were differentially expressed in more dense sperm compared to less dense sperm. For the analysis of the 11,216 mRNAs detected in sperm, we used two bioinformatics methods to measure differential gene expression; one identified 23 mRNAs that had greater concentrations in more dense sperm compared to the less dense sperm (FDR < 0.05). The second identified 163 mRNAs with greater concentrations in more dense sperm and 7 mRNAs with greater concentrations in less dense sperm (FDR < 0.05). Based on these two lists, four gene products that were of moderate to high prevalence were chosen for confirmation of differential expression by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The qRT-PCR analyses determined that SCP2 sterol-binding domain containing 1 (SCP2D1) and spermatogenesis-associated protein 31D1 (SPATA31D1) mRNA concentrations were 6- and 3-fold greater in more dense sperm, respectively, than in less dense sperm (P < 0.02). Conversely, solute carrier family 26 (anion exchanger), member 8 (SLC26A8) mRNA concentrations were 9-fold greater in less dense sperm (P < 0.05). Intriguingly, all three genes are expressed either exclusively or predominantly in sperm. Future studies will explore the functions of the products of these genes in sperm and determine their usefulness as markers of fertility.
Equine arteritis virus (EAV) is the prototype member of the family Arteriviridae, order Nidovirales. Following natural infection, 1070% of the infected stallions can become persistently infected and continue to shed EAV in semen for a variable period of time ranging from several months after infection to life-long. Therefore, carrier stallions play an important role in the maintenance of EAV in equine populations. Even though it has been determined that the EAV carrier state is testosterone-dependent, the mechanism(s) by which EAV establishes persistent infection remains to be elucidated. The objective of this study was to determine the tissue and cellular localization of EAV in the reproductive tract of long-term carrier stallions, as well as the inflammatory response induced during persistent infection. A total of 9 EAV-infected stallions (3 long-term carriers and 6 stallions that stopped viral shedding in semen) were used in this study. EAV sites of persistency were determined by virus isolation, single (EAV nucleocapsid-specific) and dual immunohistochemistry (IHC) performed on various samples collected from the reproductive tract. Furthermore, the inflammatory response was characterized by histopathology and single IHC using a panel of differentiation (CD) markers. Histological lesions were characterized by minimal to mild, focal to multifocal lymphoplasmacytic inflammation. The inflammatory response comprised moderate to high numbers of CD3+ T lymphocytes (with low to moderate, and moderate to high numbers of CD4+ and CD8+ T lymphocytes, respectively), low to moderate numbers of CD25+ T regulatory lymphocytes, minimal to moderate numbers of CD21+ B lymphocytes, and low to moderate numbers of CD83+ dendritic cells. EAV was successfully isolated from the ampulla and other accessory sex glands of carrier stallions including vesicular glands, prostate, and bulbourethral glands; and the ampulla demonstrated to be the tissue with the highest viral titer in the reproductive tract (1.0x103 e 1.7x105 PFU/g). Dual IHC demonstrated the presence of viral antigen in fibroblasts, lymphocytes (mainly CD3+ [CD8+] and CD21+), and other mononuclear cells predominantly in the lamina propria and inflammatory infiltrates in the ampulla. This study unequivocally confirms the ampulla as the primary site of EAV persistence and demonstrates that EAV has a particular tropism for fibroblasts and cells of the mononuclear lineage (including T and B lymphocytes) that participate in the inflammatory response. Interestingly, the glandular epithelium does not seem to play a role in viral maintenance during long-term persistent infection.
Acknowledgments
Key Words: EAV, carrier, tropism
Dr. G. P. Blodgett at the 6666 Ranch and Sheila Teague.
21 Sperm-bound antisperm antibodies affect motility of cooled stallion spermatozoa
Key Words: spermatozoa, RNA, fertility
20 Sites of persistent equine arteritis virus infection in the reproductive tract of the long-term carrier stallion M. Carossino 1, A.T. Loynachan 2, J.R. Campos 1, B. Nam 1, I.F. Canisso 4, Y.Y. Go 1, 3, P.J. Timoney 1, K.M. Shuck 1,
M.S. Ferrer 1, 3, L.M.J. Miller 2, 3, A. George 3, M.E. Wilkerson 3 1 Department of Large Animal Medicine, University of Georgia, Athens, GA, USA; 2 Oklahoma Equine Hospital, Washington, OK, USA; 3 College of Veterinary Medicine, Kansas State University, Manhattan, KS, USA