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

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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
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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
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Fecundity

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

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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 sperms ability to
  successfully fertilize an ovum are needed (Foote
  et al., 2003)

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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

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Fertility Differences of Bulls Bred To Cows
Synchronized With CIDRs
Yelich, 2002
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Fertility-Associated Antigen (FAA) A Unique
Seminal Protein

• FAA is one protein produced in the seminal
  vesicles, prostate, and Cowpers glands
• McCauley et al., 1999. Mol Reprod Dev 54145-153
• 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

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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
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Summary of Fertility Trials
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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)
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Bovine FAA and the Impact on Calving Season
1998 489 Head
1995 262 Head
1991 223 Head
Day
Percent Pregnant
83.8
66.0
61.4
1-30
93.4
85.1
83.4
1-45
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Immunolocalization of FAA in Multiple Species
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The FAA Gene
bFAA cDNA vs. DNase I-Like cDNA 87.5
. . . .
. 51 ACTGCTACCCTCTCTCCGAGCCCTGAAGTCA
GAG..GAGCCACGATGCCT 98
1
....................TCTTGAAGCCAGAGCAGCGCCAGGATGTCA
30 . . .
. . 99 CTGCCGCTGGCCTGCCTGCTGCTTCTC
CTCCTCTCCACCCACAGTGCCCT 148
31
CGGGAGCTGGCCCCACTGCTGCTTCTCCTCCTCTCCATCCACAGCGCCCT
80 . . .
. . 149 GGCCCTCAAGATCTGCTCCTTCAATGT
GAGGTCCTTTGGGGAATCCAAGA 198

81 GGCCATGAGGATCTGCTCCTTCAACGTCAGGTCCTTTGGGGAAAGCA
AGC 130 . . .
. . 199 AGGCAAACTGTAATGCCATGGAT
GTCATTGTGAAGGTCATCAAACGCTGT 248

131 AGGAAGACAAGAATGCCATGGATGTCATTGTGAAGGTCATCAAACG
CTGT 180 . . .
. . 249 GATATCATACTCCTGATGGAAA
TCAAGGACAGCAGCAACAGGATCTGCCC 298

181 GACATCATACTCGTGATGGAAATCAAGGACAGCAACAACAGGA
TCTGCCC 230 . .
. . . 299 CACACTGATGGAGAAGCTA
AACGGAAATTCAAGAAAAGGCATAACATACA 348

231 CATACTGATGGAGAAGCTGAACAGAAATTCAAGGAGAGGCATA
ACGTACA 280 . .
. . . 349 ACTATGTGATTAGCTCTCG
CCTTGGAAGAAACACATATAAAGAACAGTAT 398

281 ACTATGTGATTAGCTCTCGGCTTGGAAGAAACACATA
TAAAGAACAATAT 330 . .
. . . 399
GCCTTTCTCTATAAAGAAAAGCTAGTGTCTGTAAAACAAAGCTACCTCTA
448
331 GCCTTTCTCTACAAGGAAAAGCTGGT
GTCTGTGAAGAGGAGTTATCACTA 380 .
. . . . 449
CCACGACTATCAGGCTGGAGACGCAGATGTGTTTTCCAGGGAACCCTTTG
498
381 CCATGACTATCAGGATGGAGACGCAG
ATGTGTTTTCCAGGGAGCCCTTTG 430

. . . .
. 499 TGGTCTGGTTCCAGTCACCCTACACCGCTGT
CAAGGACTTCGTGATTGTC 548
431
TGGTCTGGTTCCAATCTCCCCACACTGCTGTCAAAGACTTCGTGATTATC
480 . . .
. . 549 CCCCTGCACACCACCCCTGAGACATC
CGTTAGAGAGATTGATGAGCTGGC 598

481 CCCCTGCACACCACCCCAGAGACATCCGTTAAGGAGATCG
ATGAGTTGGT 530 . .
. . . 599
TGATGTCTACACAGATGTGAAACGTCGCTGGAATGCAGAGAATTTCATTT
648
531 TGAGGTCTACACGGACGTGAAACA
CCGCTGGAAGGCGGAGAATTTCATTT 580
. . . . .
649 TCATGGGTGACTTCAATGCTGGCTGCAGCTACGTCCCCAAGAAGGC
CTGG 698
581
TCATGGGTGACTTCAATGCCGGCTGCAGCTACGTCCCCAAGAAGGCCTGG
630 . . .
. . 699 AAGGACATCCGCCTGAGGACGGACCC
CAAGTTCGTTTGGCTGATCGGGGA 748

631 AAGAACATCCGCTTGAGGACTGACCCCAGGTTTGTTTGGCTGAT
CGGGGA 680 . .
. . . 749 CCAAGAGGACACCACGGTC
AAGAAGAGCACAAACTGCGCCTATGACAGGA 798

681 CCAAGAGGACACCACGGTGAAGAAGAGCAC
CAACTGTGCATATGACAGGA 730 .
. . . . 799
TCGTGCTTAGAGGACAAAATATTGTCAACTCTGGTGGTCCTCAATCAAAC
848
731 TTGTGCTTAGAGGACAAGAAATCG
TCAGTTCTGTTGTTCCCAAGTCAAAC 780
. . . . .
849 CTCGTCTTTGATTTCCAGAAAGCTTACAGGTTGTCTGAATCGAAGG
CCCT 898
781 AGTGTTTTTGACTTCCAGAAA
GCTTACAAGCTGACTGAAGAGGAGGCCCT 830
. . . . .
899 GG............................................
.... 900
831 GG
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Isolate
ATGGGTGAC
mRNA
TTCTAA

.
cDNA
Perform RT
-
PCR
Isolate bacteria
and Purify
Protein
Sequence
Propagate
..ATGGGCCATAGA.CAT
CATCATCATCATCAT
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Development of a Chute-Side Kit
Western Blot
Semen Sample
Test Result

Control
Unknown
Available from ReproTec, Inc. Tucson, AZ
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FAA Status in Commercial Herds in 6 States
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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
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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 lt 0.05).
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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

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Motility and percent intact acrosomes (PIA) of
gender-sorted sperm 3 h post-thaw.
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Neutrophil Extracellular Traps (NETs)
Alghamdi and Foster, 2005
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rbFAA Inhibits Sperm-Neutrophil Binding
Percentage change from control
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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.

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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

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