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A 25-Year Chronology of a Seminal Protein: Diagnostic and Therapeutic Utility

A 25-Year Chronology of a Seminal Protein: Diagnostic and Therapeutic Utility. Roy L. Ax, Tod C. McCauley, and Kelsey E. Shupe. Department of Animal Sciences, University of Arizona TMI Laboratories International, Inc. Tucson, AZ. Breeding Soundness Qualification.

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A 25-Year Chronology of a Seminal Protein: Diagnostic and Therapeutic Utility

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  1. A 25-Year Chronology of a Seminal Protein: Diagnostic and Therapeutic Utility Roy L. Ax, Tod C. McCauley, and Kelsey E. Shupe Department of Animal Sciences, University of Arizona TMI Laboratories International, Inc. Tucson, AZ

  2. Breeding Soundness Qualification • Visual assessment of feet, legs, eyes, teeth, external genitalia; i.e., assessment of mating ability • Palpation of accessory sex glands • Scrotal measurement, evaluation of testis, epididymis • Evaluation of a semen sample

  3. Semen Characteristics • Concentration • Motility • Morphology

  4. Bull Fertility • Abnormal forms of sperm have the highest relationship to fertility assessing a fresh ejaculate • % Intact acrosomes (PIA) 3h post-thaw has a 92% correlation with A.I. fertility when evaluating frozen/thawed specimens

  5. Semen testing among all cow-calf operations (1997) NAHMS population study

  6. Fecundity • From DNA parentage verification, 20% of bulls in natural service breeding will sire 80% of the offspring

  7. Identification of the Problem: • Males who are subjected to a fertility exam, and pass, still vary dramatically in actual fertility. • In mammalian males, objective biochemical markers which are indicators of a sperm’s ability to successfully fertilize an ovum are needed (Foote et al., 2003)

  8. FACT: Freshly ejaculated mammalian sperm cannot fertilize an egg • In the female, over a 6-8h timespan, a process called capacitation occurs • Sperm acquire the capacity to fertilize

  9. Fertility Differences of Bulls Bred To Cows Synchronized With CIDRs Yelich, 2002

  10. Fertility-Associated Antigen (FAA): A Unique Seminal Protein • FAA is one protein produced in the seminal vesicles, prostate, and Cowper’s glands • McCauley et al., 1999. Mol Reprod Dev; 54:145-153 • FAA functions as a docking molecule so female secretions can cause capacitation. • Serves as a biomarker for fertility potential of bulls • Therefore, FAA plays a pivotal role in sperm meets egg likelihood

  11. Diagnostic Test Gel Electrophoresis Western Blot Large Protein Size (kDa) 31 Transfer proteins Incubate with antibodies Proteins removed from sperm 27 24 Small Proteins separated by electrical current Only proteins that bind antibody are identified

  12. Summary of Fertility Trials

  13. Fertility of Range and A.I. Bulls Based on FAA Status 16.1 % (Data adapted from Bellin et al., 1994, 96,98; Sprott et al., 2000; and unpublished data)

  14. Bovine FAA and the Impact on Calving Season 1991* 223 Head 1995 262 Head 1998 489 Head Day Percent Pregnant 1-30 61.4 66.0 83.8 1-45 83.4 85.1 93.4

  15. Immunolocalization of FAA in Multiple Species

  16. The FAA Gene bFAA cDNA vs. DNase I-Like cDNA: 87.5% . . . . . 51 ACTGCTACCCTCTCTCCGAGCCCTGAAGTCAGAG..GAGCCACGATGCCT 98 | ||||| ||||| | |||| |||| | 1 ....................TCTTGAAGCCAGAGCAGCGCCAGGATGTCA 30 . . . . . 99 CTGCCGCTGGCCTGCCTGCTGCTTCTCCTCCTCTCCACCCACAGTGCCCT 148 | | ||||||| |||||||||||||||||||||| |||||| ||||| 31 CGGGAGCTGGCCCCACTGCTGCTTCTCCTCCTCTCCATCCACAGCGCCCT 80 . . . . . 149 GGCCCTCAAGATCTGCTCCTTCAATGTGAGGTCCTTTGGGGAATCCAAGA 198 |||| | | ||||||||||||||| || ||||||||||||||| |||| 81 GGCCATGAGGATCTGCTCCTTCAACGTCAGGTCCTTTGGGGAAAGCAAGC 130 . . . . . 199 AGGCAAACTGTAATGCCATGGATGTCATTGTGAAGGTCATCAAACGCTGT 248 ||| | || ||||||||||||||||||||||||||||||||||||||| 131 AGGAAGACAAGAATGCCATGGATGTCATTGTGAAGGTCATCAAACGCTGT 180 . . . . . 249 GATATCATACTCCTGATGGAAATCAAGGACAGCAGCAACAGGATCTGCCC 298 || ||||||||| ||||||||||||||||||||| ||||||||||||||| 181 GACATCATACTCGTGATGGAAATCAAGGACAGCAACAACAGGATCTGCCC 230 . . . . . 299 CACACTGATGGAGAAGCTAAACGGAAATTCAAGAAAAGGCATAACATACA 348 || ||||||||||||||| ||| |||||||||| | ||||||||| |||| 231 CATACTGATGGAGAAGCTGAACAGAAATTCAAGGAGAGGCATAACGTACA 280 . . . . . 349 ACTATGTGATTAGCTCTCGCCTTGGAAGAAACACATATAAAGAACAGTAT 398 ||||||||||||||||||| |||||||||||||||||||||||||| ||| 281 ACTATGTGATTAGCTCTCGGCTTGGAAGAAACACATATAAAGAACAATAT 330 . . . . . 399 GCCTTTCTCTATAAAGAAAAGCTAGTGTCTGTAAAACAAAGCTACCTCTA 448 ||||||||||| || |||||||| |||||||| || || || | ||| 331 GCCTTTCTCTACAAGGAAAAGCTGGTGTCTGTGAAGAGGAGTTATCACTA 380 . . . . . 449 CCACGACTATCAGGCTGGAGACGCAGATGTGTTTTCCAGGGAACCCTTTG 498 ||| |||||||||| ||||||||||||||||||||||||||| ||||||| 381 CCATGACTATCAGGATGGAGACGCAGATGTGTTTTCCAGGGAGCCCTTTG 430 . . . . . 499 TGGTCTGGTTCCAGTCACCCTACACCGCTGTCAAGGACTTCGTGATTGTC 548 ||||||||||||| || ||| |||| |||||||| |||||||||||| || 431 TGGTCTGGTTCCAATCTCCCCACACTGCTGTCAAAGACTTCGTGATTATC480 . . . . . 549CCCCTGCACACCACCCCTGAGACATCCGTTAGAGAGATTGATGAGCTGGC598 ||||||||||||||||| ||||||||||||| ||||| |||||| ||| 481CCCCTGCACACCACCCCAGAGACATCCGTTAAGGAGATCGATGAGTTGGT530 . . . . . 599TGATGTCTACACAGATGTGAAACGTCGCTGGAATGCAGAGAATTTCATTT648 ||| |||||||| || ||||||| |||||||| || ||||||||||||| 531TGAGGTCTACACGGACGTGAAACACCGCTGGAAGGCGGAGAATTTCATTT580 . . . . . 649TCATGGGTGACTTCAATGCTGGCTGCAGCTACGTCCCCAAGAAGGCCTGG 698 ||||||||||||||||||| |||||||||||||||||||||||||||||| 581TCATGGGTGACTTCAATGCCGGCTGCAGCTACGTCCCCAAGAAGGCCTGG 630 . . . . . 699AAGGACATCCGCCTGAGGACGGACCCCAAGTTCGTTTGGCTGATCGGGGA748 ||| |||||||| ||||||| ||||||| ||| ||||||||||||||||| 631AAGAACATCCGCTTGAGGACTGACCCCAGGTTTGTTTGGCTGATCGGGGA680 . . . . . 749CCAAGAGGACACCACGGTCAAGAAGAGCACAAACTGCGCCTATGACAGGA 798 |||||||||||||||||| ||||||||||| ||||| || |||||||||| 681CCAAGAGGACACCACGGTGAAGAAGAGCACCAACTGTGCATATGACAGGA 730 . . . . . 799TCGTGCTTAGAGGACAAAATATTGTCAACTCTGGTGGTCCTCAATCAAAC 848 | ||||||||||||||| | || |||| |||| || ||| | |||||| 731TTGTGCTTAGAGGACAAGAAATCGTCAGTTCTGTTGTTCCCAAGTCAAAC780 . . . . . 849CTCGTCTTTGATTTCCAGAAAGCTTACAGGTTGTCTGAATCGAAGGCCCT898 || ||||| |||||||||||||||| | || ||||| | ||||||| 781AGTGTTTTTGACTTCCAGAAAGCTTACAAGCTGACTGAAGAGGAGGCCCT830 . . . . . 899 GG................................................ 900 || 831 GG

  17. Producing a Recombinant Protein: Producing a Recombinant Protein: Producing a Recombinant Protein: Isolate ATGGGTGAC mRNA TTCTAA …… . cDNA Harvest Tissue Harvest Tissue Harvest Tissue Perform RT - PCR Isolate bacteria and Purify Protein Sequence Propagate …..ATGGGCCATAGA…….CAT CATCATCATCATCAT Isolate ATGGGTGAC mRNA TTCTAA …… . cDNA Perform RT - PCR Isolate bacteria and Purify Protein Sequence Propagate …..ATGGGCCATAGA…….CAT CATCATCATCATCAT

  18. Development of a Chute-Side Kit Western Blot Test Result Semen Sample Control Unknown Available from ReproTec, Inc. Tucson, AZ

  19. FAA Status in Commercial Herds in 6 States

  20. FAA Negative Yearlings FAA Negative Yearlings Pedigree of FAA Negative Bulls With a Common Ancestor X Sire A In a subset of 22 FAA negative sires, 18 were sired by A or B and all had bull X in their pedigree Dam Gr Sire X Sire B Sire A Gr Dam Dam

  21. Recombinant FAA potentiated heparin-induced capacitation of bovine sperm in vitro Data were analyzed by ANOVA. Least squares means and SEM are shown. Values without a common superscript differ (P < 0.05).

  22. The percentage change in intact acrosomes during a 3 h incubation post-thaw. Supplementation with rFAA and rTIMP-2 Increased the proportion of sperm with intact acrosomes by an average of 1.8% while control samples decreased an average of 7.1 %

  23. Motility and percent intact acrosomes (PIA) of gender-sorted sperm 3 h post-thaw.

  24. Neutrophil Extracellular Traps (NETs) Alghamdi and Foster, 2005

  25. rbFAA Inhibits Sperm-Neutrophil Binding Percentage change from control

  26. Summary • A rapid chute-side test for FAA is commercially available • Recombinant forms of FAA (and TIMP-2) have been produced. • Those recombinant proteins potentiate heparin-induced capacitation and prevent loss of motility and acrosome integrity during cryopreservation. • DNA mutation have been identified in the FAA gene. • Those mutations may prove to be useful diagnostic markers for fertility.

  27. WHAT WE NOW KNOW ABOUT FAA • Its presence on sperm is a biomarker for fertility • Addition to semen potentiates capacitation of sperm • Addition to semen improves sperm function post-thaw (improves motility and % intact acrosomes) • Reduces sperm-neutrophil binding in dose-dependent manner

  28. Acknowledgements M.E. Bellin Dr. G.R. Dawson Amy Perkins Dr. S.H.F. Marks H.E. Hawkins Dr. H.M. Zhang Dr. R.W. Lenz J.N. Oyarzo Dr. L.R. Sprott

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