1. By:By:
Dr. Subodh KumarDr. Subodh Kumar
Senior Scientist,Senior Scientist,
Division of Animal GeneticsDivision of Animal Genetics
Indian Veterinary Research Institute, IzatnagarIndian Veterinary Research Institute, Izatnagar

2. OutlayOutlay
– Historical Background - need for research ?Historical Background - need for research ?
– Platelet activating factor (PAF)Platelet activating factor (PAF)
• IntroductionIntroduction
• Chemical and structural formulaeChemical and structural formulae
• SignificanceSignificance
• Synthesis and metabolism.Synthesis and metabolism.
– Role of PAF in reproductive biologyRole of PAF in reproductive biology
• Female reproductionFemale reproduction
– OvulationOvulation
– FertilizationFertilization
– Embryo cleavageEmbryo cleavage
– ImplantationImplantation
– PregnancyPregnancy
– ParturitionParturition
• Male reproductionMale reproduction
– Sperm motilitySperm motility
– Acrosome reactionAcrosome reaction
– Brief reports of the work done in LabBrief reports of the work done in Lab

3. Name assigned to Glycerophospholipids.Name assigned to Glycerophospholipids.
Identified in several reproductive tissues.Identified in several reproductive tissues.
Human / mouse pre-implantation embryosHuman / mouse pre-implantation embryos
produce and secrete PAF into culture media inproduce and secrete PAF into culture media in
vitro.vitro.
Embryo derived PAF – mild thrombocytopeniaEmbryo derived PAF – mild thrombocytopenia
in women/mouse receiving IVF-ET embryos.in women/mouse receiving IVF-ET embryos.
PAF involved with follicular rupture duringPAF involved with follicular rupture during
ovulation in rats.ovulation in rats.

4. Synthetic PAF has shown biological activitiesSynthetic PAF has shown biological activities
indistinguishable from naturally occurring PAFindistinguishable from naturally occurring PAF
(Demopoulous(Demopoulous et alet al, 1979)., 1979).
One of the most potent secondary inflammationOne of the most potent secondary inflammation
mediators in mammalian tissues (Braquetmediators in mammalian tissues (Braquet et alet al,,
1987).1987).
Synthesised via remodelling pathway (lyso PAFSynthesised via remodelling pathway (lyso PAF
being the immediate precursor and metabolite ofbeing the immediate precursor and metabolite of
PAF) at the place and time when the activity isPAF) at the place and time when the activity is
requiredrequired..
Acetyl HydrolaseAcetyl Hydrolase
Lyso PAFLyso PAF PAFPAF
Acetyl TransferaseAcetyl Transferase

5. IUPAC Nomenclature of PAF:
1-o-alkyl-2-acetyl-sn-glycero-3-phosphoryl
chorine
Structural formulae of PAF:
O H2
1
C O (CH2)15-17* CH3
CH3 C O 2
C H O CH3
H2
3
C O P O CH2 CH2
+
N CH3
O -
CH3
* Side Chain is mainly C 18:0 or C 16:0 compound

6. Role of PAF in female reproductive biology.Role of PAF in female reproductive biology.
(i) Ovulation and luteal function(i) Ovulation and luteal function
PAF participates in inflammation process hence its rolePAF participates in inflammation process hence its role
in ovulation has been suggested. Concentration ofin ovulation has been suggested. Concentration of
ovarian PAF decreased by 36 % after 2 hrs of hCGovarian PAF decreased by 36 % after 2 hrs of hCG
treatment (Espeytreatment (Espey et alet al, 1989)., 1989).
PAF specific antagonist BN 52021 inhibited the folliclePAF specific antagonist BN 52021 inhibited the follicle
rupture in rats, which were stimulated to ovulate withrupture in rats, which were stimulated to ovulate with
hCG. Action reversed by simultaneous administration ofhCG. Action reversed by simultaneous administration of
PAF (AbisogunPAF (Abisogun et alet al, 1989)., 1989).
PAF was produced by the embryos at the time of corpusPAF was produced by the embryos at the time of corpus
luteum formation. Synthetic PAF at 100 -1000 ng/ml ofluteum formation. Synthetic PAF at 100 -1000 ng/ml of
medium stimulated the enhancement of progesteronemedium stimulated the enhancement of progesterone
production by human granulose cell in culture (O’Neill,production by human granulose cell in culture (O’Neill,
1987).1987).

7. (ii) Fertilization and embryo cleavage.(ii) Fertilization and embryo cleavage.
PAF can induce two fold increase in fertilization rates inPAF can induce two fold increase in fertilization rates in
mouse oocytes in vitro at nano molar to micro molarmouse oocytes in vitro at nano molar to micro molar
concentration within a very short interaction time betweenconcentration within a very short interaction time between
the two gametes, whereas the presence of PAF antagonistthe two gametes, whereas the presence of PAF antagonist
CV-3988 caused significant decrease in fertilization ratesCV-3988 caused significant decrease in fertilization rates
(Minhas(Minhas et alet al, 1989)., 1989).
Supporting studies: Two - three fold increase inSupporting studies: Two - three fold increase in
fertilization rates in mouse oocytes which were decreased byfertilization rates in mouse oocytes which were decreased by
PAF antagonist WEB-2086 (ShiPAF antagonist WEB-2086 (Shi et alet al, 1992)., 1992).
PAF concentrations increased with the advancing stages ofPAF concentrations increased with the advancing stages of
development of murine embryos thus suggesting its role indevelopment of murine embryos thus suggesting its role in
embryo cleavage (Rippsembryo cleavage (Ripps et alet al, 1993)., 1993).
Supporting studies: Significantly higher mitotic index ofSupporting studies: Significantly higher mitotic index of
mouse blastocysts with PAF.mouse blastocysts with PAF.
Recent report- IVF rates were increased when PAF wasRecent report- IVF rates were increased when PAF was
incorporated in capacitation and fertilization medium inincorporated in capacitation and fertilization medium in
murine embryos (Sarvanan and Sharma, 1997).murine embryos (Sarvanan and Sharma, 1997).

8. (iii) Implantation(iii) Implantation
Embryo derived PAF has been postulated to be the signalEmbryo derived PAF has been postulated to be the signal
required to induce EPF during the implantation stage ofrequired to induce EPF during the implantation stage of
pregnancy (Orozcopregnancy (Orozco et alet al, 1986)., 1986).
PAF antagonist viz. iloprost, SRI–63-441 is known to causePAF antagonist viz. iloprost, SRI–63-441 is known to cause
inhibition of implantation of mouse embryos which couldinhibition of implantation of mouse embryos which could
be reversed by simultaneous administration of PAF-be reversed by simultaneous administration of PAF-
indicates the necessity of PAF for successful implantation.indicates the necessity of PAF for successful implantation.
(Spinkes and O’Neill, 1988).(Spinkes and O’Neill, 1988).
Intra-uterine administration of PAF antagonist BN 52021Intra-uterine administration of PAF antagonist BN 52021
was known to inhibit the ovo-implantation in rats in a dosewas known to inhibit the ovo-implantation in rats in a dose
dependent fashion (Ackerdependent fashion (Acker et alet al, 1988)., 1988).
Two cell mice embryos cultured in medium with PAF hadTwo cell mice embryos cultured in medium with PAF had
a higher in vitro implantation rate as compared to controls.a higher in vitro implantation rate as compared to controls.
Also enhanced theAlso enhanced the in vitroin vitro implantation rate in later stagesimplantation rate in later stages
of embryo development (Nishiof embryo development (Nishi et alet al, 1995)., 1995).

9. (iv) Pregnancy and Parturition(iv) Pregnancy and Parturition
PAF is known to be produced by human embryos after IVFPAF is known to be produced by human embryos after IVF
and embryo transfer – one of the earliest signals for theand embryo transfer – one of the earliest signals for the
maternal recognition of pregnancy (O’Neill, 1987).maternal recognition of pregnancy (O’Neill, 1987).
PAF may play a role in foetal lung maturation as after intraPAF may play a role in foetal lung maturation as after intra
peritoneal administration into foetus, it caused decrease inperitoneal administration into foetus, it caused decrease in
foetal pulmonary and hepatic glycogen concentration withfoetal pulmonary and hepatic glycogen concentration with
concomitant increase in pulmonary and hepatic plasmaconcomitant increase in pulmonary and hepatic plasma
lactate levels.lactate levels.
PAF present in the amniotic fluid of the pregnant womenPAF present in the amniotic fluid of the pregnant women
with a concentration increasing mid gestation onwards andwith a concentration increasing mid gestation onwards and
becoming very high just before the onset of labor, thusbecoming very high just before the onset of labor, thus
suggesting the role of PAF in parturition (Bernalsuggesting the role of PAF in parturition (Bernal et alet al, 1991)., 1991).
PAF receptor antagonist 1-659, 989 are known to prolong thePAF receptor antagonist 1-659, 989 are known to prolong the
duration of parturition by 2-5 folds upon administration induration of parturition by 2-5 folds upon administration in
pregnant rats.pregnant rats.
PAF receptor antagonist interfere in the normal progressionPAF receptor antagonist interfere in the normal progression
of events in parturition and PAF may be one of the mediatersof events in parturition and PAF may be one of the mediaters
for the initiation of myometrical contractions necessary forfor the initiation of myometrical contractions necessary for
the expulsion of foetus (Yan-Pig Zhuthe expulsion of foetus (Yan-Pig Zhu et alet al, 1991)., 1991).

10. Role of PAF in male reproductive biology.Role of PAF in male reproductive biology.
Sperm motility and acrosome reaction (AR).Sperm motility and acrosome reaction (AR).
PAF acetyl hydrolase activity (which inactivates the PAF) wasPAF acetyl hydrolase activity (which inactivates the PAF) was
found to be present in human, bovine, hamster and rabbitfound to be present in human, bovine, hamster and rabbit
seminal plasma (Hough and Parks, 1994).seminal plasma (Hough and Parks, 1994).
PAF increases the mean swimming speed of humanPAF increases the mean swimming speed of human
spermatozoa at nano molar concentrations (Rickerspermatozoa at nano molar concentrations (Ricker et alet al, 1989)., 1989).
At micro molar concentration, it may decrease the motility andAt micro molar concentration, it may decrease the motility and
induce AR in human spermatozoa (D’Cruz and Haas, 1989;induce AR in human spermatozoa (D’Cruz and Haas, 1989;
RickerRicker et alet al, 1989)., 1989).
Spermatozoa undergo AR without rapid loss of motility atSpermatozoa undergo AR without rapid loss of motility at
micromolar concentration in cattlemicromolar concentration in cattle ..
 CattleCattle : Parks and Hough, 1990: Parks and Hough, 1990
 Cattle and Buffalo: Aravindakshan and Sharma, 1995,1996;Cattle and Buffalo: Aravindakshan and Sharma, 1995,1996;
Kumar and Sharma, 2001.Kumar and Sharma, 2001.
 MouseMouse : Sarvanan and Sharma,1997.: Sarvanan and Sharma,1997.

11. Possible mechanism of PAF in Capacitation and AR ofPossible mechanism of PAF in Capacitation and AR of
spermatozoaspermatozoa
PAF binds to target cellsPAF binds to target cells
Increase in inositol 4-5 bi phosphate breakdownIncrease in inositol 4-5 bi phosphate breakdown
Di acyl glycerolDi acyl glycerol
Generation of inositol 1,4,5 tri phosphateGeneration of inositol 1,4,5 tri phosphate
Calcium mobilization increasedCalcium mobilization increased
Membrane fusionMembrane fusion Elevation ofElevation of
by lysophospholipidsby lysophospholipids Intracellular calciumIntracellular calcium
Capacitation and Acrosome reactionCapacitation and Acrosome reaction

12. Values based on 40 ejaculatesValues based on 40 ejaculates
Effect of PAF on Motility of Karan Fries spermatozoa
(fresh)
0
20
40
60
80
0 15 30 60 120
Duration of incubation (min)
Motility(%)
Control
50
100
150
200

13. Values based on 25 ejaculatesValues based on 25 ejaculates
Effect of PAF on Motility of Karan Swiss spermatozoa
(fresh)
0
20
40
60
80
0 15 30 60 120
Duration of incubation (min)
Motility(%)
Control
50
100
150
200

14. Values based on 25 ejaculatesValues based on 25 ejaculates
Effect of PAF on Motility of Murrah spermatozoa (fresh)
0
10
20
30
40
50
60
70
80
0 15 30 60 120
Duration of incubation (min)
Motility(%)
Control
50
100
150
200

15. Values based on 40 ejaculatesValues based on 40 ejaculates
Effect of PAF on Acrosome Reaction of Karan Fries
spermatozoa (fresh)
0
20
40
60
80
100
120
0 15 30 60 120
Duration of incubation (min)
AcrosomrReaction
(%)
Control
50
100
150
200

16. Values based on 25 ejaculatesValues based on 25 ejaculates
Effect of PAF on Acrosome Reaction of Karan Swiss
spermatozoa (fresh)
0
20
40
60
80
100
120
0 15 30 60 120
Duration of incubation (min)
AcrosomeReaction
(%)
Control
50
100
150
200

17. Values based on 25 ejaculatesValues based on 25 ejaculates
Effect of PAF on Acrosome Reaction of Murrah
spermatozoa (fresh)
0
20
40
60
80
100
120
0 15 30 60 120
Duration of incubation (min)
AcrosomeReaction
(%)
Control
50
100
150
200

18. Effect of 100 M PAF omMotilityand Acrosome
Reaction of Karan Fries spermatozoa (fresh)
0
20
40
60
80
0 15 30 60 120
Duration of incubation (min)
PercentageValue
MOT
AR

19. Effect of 100 UM PAF on Motilityand Acrosome
Reaction of Karan Swiss spermatozoa (fresh)
0
20
40
60
80
0 15 30 60 120
Duration of incubation (min)
ParcentageValue
MOT
AR

20. Effect of 100 UM PAF on Motility and Acrosome
Reaction of Murrah spermatozoa (fresh)
0
20
40
60
80
0 15 30 60 120
Duration of incubation (min)
PaercentageValue
MOT
AR

21. Relative changes in percent motility and acrosome reactionRelative changes in percent motility and acrosome reaction
in Karan Swiss - fresh and frozen spermatozoa at the end ofin Karan Swiss - fresh and frozen spermatozoa at the end of
15 and 30 min of incubation (at 10015 and 30 min of incubation (at 100 µµM of PAF).M of PAF).
CharacterCharacter SemenSemen
TypeType
Time of ReactionTime of Reaction % change% change
from 0 tofrom 0 to
15 min15 min
% change% change
from 15 tofrom 15 to
30 min30 min
0 min0 min 15 min15 min 30 min30 min
MotilityMotility
(mean(mean ±± SE)SE)
FreshFresh 71.871.8 ±±
1.051.05
61.261.2 ±±
1.481.48
55.855.8 ±±
1.691.69
-14.76-14.76 -8.82-8.82
FrozenFrozen 51.351.3 ±±
1.911.91
45.745.7 ±±
1.551.55
41.741.7 ±±
1.741.74
-10.92-10.92 -8.75-8.75
ARAR
(mean(mean ±± SE)SE)
FreshFresh 4.74.7 ±± 0.510.51 45.045.0 ±±
2.622.62
48.148.1 ±±
2.672.67
+857.45+857.45 +6.89+6.89
FrozenFrozen 18.918.9 ±±
2.792.79
33.433.4 ±±
3.603.60
34.634.6 ±±
3.433.43
+43.41+43.41 +3.59+3.59

22. Relative changes in percent motility and acrosome reactionRelative changes in percent motility and acrosome reaction
in Karan Fries- fresh and frozen spermatozoa at the end ofin Karan Fries- fresh and frozen spermatozoa at the end of
15 and 30 min of incubation (at 10015 and 30 min of incubation (at 100 µµM of PAF).M of PAF).
CharacterCharacter SemenSemen
TypeType
Time of ReactionTime of Reaction % change% change
from 0 tofrom 0 to
15 min15 min
% change% change
from 15 tofrom 15 to
30 min30 min
0 min0 min 15 min15 min 30 min30 min
MotilityMotility
(mean(mean ±± SE)SE)
FreshFresh 72.672.6 ±±
0.790.79
62.962.9 ±±
1.121.12
58.658.6 ±±
1.181.18
-13.36-13.36 -6.84-6.84
FrozenFrozen 45.345.3 ±±
1.731.73
39.339.3 ±±
2.072.07
34.334.3 ±±
1.991.99
-13.25-13.25 -12.72-12.72
ARAR
(mean(mean ±± SE)SE)
FreshFresh 3.63.6 ±± 0.250.25 45.645.6 ±±
1.751.75
46.446.4 ±±
1.051.05
+1166.67+1166.67 +1.75+1.75
FrozenFrozen 19.519.5 ±±
2.512.51
37.737.7 ±±
1.841.84
39.039.0 ±±
2.272.27
+93.33+93.33 +3.45+3.45

23. Relative changes in percent motility and acrosome reactionRelative changes in percent motility and acrosome reaction
in Murrah- fresh and frozen spermatozoa at the end of 15in Murrah- fresh and frozen spermatozoa at the end of 15
and 30 min of incubation (at 100and 30 min of incubation (at 100 µµM of PAF).M of PAF).
CharacterCharacter SemenSemen
TypeType
Time of ReactionTime of Reaction % change% change
from 0 tofrom 0 to
15 min15 min
% change% change
from 15 tofrom 15 to
30 min30 min
0 min0 min 15 min15 min 30 min30 min
MotilityMotility
(mean(mean ±± SE)SE)
FreshFresh 72.672.6 ±±
1.241.24
60.060.0 ±±
1.671.67
54.854.8 ±±
1.461.46
-17.36-17.36 -8.67-8.67
FrozenFrozen 56.356.3 ±±
1.531.53
52.352.3 ±±
1.551.55
46.746.7 ±±
1.611.61
-7.11-7.11 -10.71-10.71
ARAR
(mean(mean ±± SE)SE)
FreshFresh 4.44.4 ±±0.630.63 34.234.2 ±±
1.411.41
37.537.5 ±±
1.351.35
+677.27+677.27 +9.65+9.65
FrozenFrozen 17.517.5 ±±
2.842.84
27.327.3 ±±
1.841.84
28.928.9 ±±
2.052.05
+56.00+56.00 +5.66+5.66

24. EFFECT OF PAF ON HYALURONIDASE ACTIVITY IN
KARAN FRIES SEMEN
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0.16
1 2 3 4 5 6 7 8 9 10 11 12
BULL NUMBER
HYALURONIDASEACTIVITY(UNITS)
CONTROLS
TREATED

25. EFFECT OF PAF ON HYALURONIDASE ACTIVITY IN
MURRAH SEMEN
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0.16
1 2 3 4 5 6 7 8 9 10 11 12
BULL NUMBER
HYALURONIDASEACTIVITY(UNITS)
CONTROLS
TREATED

26. EFFECT OF PAF ON FERTILIZATION INDEX IN
KARAN FRIES BULLS
0
0.2
0.4
0.6
0.8
1
1.2
1 2 3 4 5 6 7 8 9 10 11 12
BULL NUMBER
FERTILIZATIONINDEX
CONTROLS
TREATED

27. EFFECT OF PAF ON FERTILIZATION INDEX IN
MURRAH BULLS
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1 2 3 4 5 6 7 8 9 10 11 12
BULL NUMBER
FERTILIZATIONINDEX
CONTROLS
TREATED

28. Rate of in vitro fertilization in different group of experiments.
Trial # 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Total
A I 9 11 38 24 22 9 15 23 24 27 16 7 14 22 16 34 11 10 23 22 377
F 2 2 5 7 8 5 6 7 3 13 4 1 4 2 3 6 4 3 5 3 93
% 22 18 13 29 36 55 40 30 12 48 25 14 28 9 19 17 36 30 21 13 24.9 ±
0.10
B I 42 17 65 17 20 21 22 20 32 30 11 22 39 39 13 27 21 13 51 11 533
F 7 7 20 9 6 4 7 6 4 16 6 4 5 3 1 6 5 9 10 3 140
% 16 52 30 52 30 19 31 30 12 53 54 18 12 7 7 22 23 69 19 27 27.7 ±
0.20
C I 8 16 18 15 24 16 20 16 20 9 12 15 26 7 21 18 11 10 39 26 347
F 3 6 7 6 9 7 4 6 3 2 6 5 3 4 2 3 4 4 4 7 95
% 37 37 38 40 31 43 20 37 15 22 50 33 11 57 9 16 36 40 10 26 29.4 ±
0.13
D I 18 9 16 8 39 17 18 17 22 20 9 16 33 37 14 11 10 11 15 12 352
F 5 2 4 3 8 6 4 7 7 3 3 4 8 11 4 6 4 5 6 3 103
% 27 22 25 37 20 35 22 41 31 15 33 25 24 29 28 54 40 45 40 25 30.5 ±
0.06

29. Rate of in vitro fertilization in different group of experiments.Rate of in vitro fertilization in different group of experiments.
• I= No. of oocytes inseminatedI= No. of oocytes inseminated
• F = No. of oocytes fertilizedF = No. of oocytes fertilized
• % = Percentage of fertilization% = Percentage of fertilization
• A= Sperm control and no PAF treatment to IVF and IVCA= Sperm control and no PAF treatment to IVF and IVC
• B= Sperm control and PAF treatment to IVF and IVCB= Sperm control and PAF treatment to IVF and IVC
• C= Sperm treated and no PAF treatment to IVF and IVCC= Sperm treated and no PAF treatment to IVF and IVC
• D= Sperm treated and PAF treatment to IVF and IVCD= Sperm treated and PAF treatment to IVF and IVC
• Effect of addition of PAF to sperm =Changes in fertilization rate fromEffect of addition of PAF to sperm =Changes in fertilization rate from
A to C = 18.0 %A to C = 18.0 %
• Effect of addition of PAF to IVF medium =Changes in fertilizationEffect of addition of PAF to IVF medium =Changes in fertilization
rate from A to B = 11.2 %rate from A to B = 11.2 %
• Effect of addition of PAF to sperm and IVF medium =Changes inEffect of addition of PAF to sperm and IVF medium =Changes in
fertilization rate from A to D = 22.48 %fertilization rate from A to D = 22.48 %

30. A= Sperm control and no PAF treatment to IVF and IVCA= Sperm control and no PAF treatment to IVF and IVC
B= Sperm control and PAF treatment to IVF and IVCB= Sperm control and PAF treatment to IVF and IVC
C= Sperm treated and no PAF treatment to IVF and IVCC= Sperm treated and no PAF treatment to IVF and IVC
D= Sperm treated and PAF treatment to IVF and IVCD= Sperm treated and PAF treatment to IVF and IVC
Culture and Development of Mouse Embryos
0
20
40
60
80
100
2-CELL 4-CELL 8-CELL 16-CELL MORULLA
DevelopmentRate(%)
A
B
C
D

31. ConclusionConclusion