platelet activating factor
TRANSCRIPT
By: By:
Dr. Subodh KumarDr. Subodh KumarSenior Scientist,Senior Scientist,
Division of Animal GeneticsDivision of Animal GeneticsIndian Veterinary Research Institute, IzatnagarIndian Veterinary Research Institute, Izatnagar
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
Name assigned to Glycerophospholipids.Name assigned to Glycerophospholipids.
Identified in several reproductive tissues.Identified in several reproductive tissues.
Human / mouse pre-implantation embryos Human / mouse pre-implantation embryos
produce and secrete PAF into culture media in produce 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.
Synthetic PAF has shown biological activities Synthetic PAF has shown biological activities indistinguishable from naturally occurring PAF indistinguishable from naturally occurring PAF (Demopoulous (Demopoulous et alet al, 1979)., 1979).
One of the most potent secondary inflammation One of the most potent secondary inflammation mediators in mammalian tissues (Braquet mediators in mammalian tissues (Braquet et alet al, , 1987).1987).
Synthesised via remodelling pathway (lyso PAF Synthesised via remodelling pathway (lyso PAF being the immediate precursor and metabolite of being the immediate precursor and metabolite of PAF) at the place and time when the activity is PAF) at the place and time when the activity is requiredrequired. .
Acetyl HydrolaseAcetyl Hydrolase
Lyso PAF Lyso PAF PAFPAF
Acetyl TransferaseAcetyl Transferase
IUPAC Nomenclature of PAF:
1-o-alkyl-2-acetyl-sn-glycero-3-phosphoryl chorine
Structural formulae of PAF:
O H2 1C O (CH2)15-17* CH3
CH3 C O 2C H O CH3
H2 3C O P O CH2 CH2 +N CH3
O - CH3
* Side Chain is mainly C 18:0 or C 16:0 compound* Side Chain is mainly C 18:0 or C 16:0 compound
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 role PAF participates in inflammation process hence its role in ovulation has been suggested. Concentration of in ovulation has been suggested. Concentration of ovarian PAF decreased by 36 % after 2 hrs of hCG ovarian PAF decreased by 36 % after 2 hrs of hCG treatment (Espey treatment (Espey et alet al, 1989)., 1989).
PAF specific antagonist BN 52021 inhibited the follicle PAF specific antagonist BN 52021 inhibited the follicle rupture in rats, which were stimulated to ovulate with rupture in rats, which were stimulated to ovulate with hCG. Action reversed by simultaneous administration of hCG. Action reversed by simultaneous administration of PAF (Abisogun PAF (Abisogun et alet al, 1989)., 1989).
PAF was produced by the embryos at the time of corpus PAF was produced by the embryos at the time of corpus luteum formation. Synthetic PAF at 100 -1000 ng/ml of luteum formation. Synthetic PAF at 100 -1000 ng/ml of medium stimulated the enhancement of progesterone medium 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).
(ii) Fertilization and embryo cleavage.(ii) Fertilization and embryo cleavage.PAF can induce two fold increase in fertilization rates in PAF can induce two fold increase in fertilization rates in mouse oocytes in vitro at nano molar to micro molar mouse oocytes in vitro at nano molar to micro molar concentration within a very short interaction time between concentration within a very short interaction time between the two gametes, whereas the presence of PAF antagonist the two gametes, whereas the presence of PAF antagonist CV-3988 caused significant decrease in fertilization rates CV-3988 caused significant decrease in fertilization rates (Minhas (Minhas et alet al, 1989)., 1989).
Supporting studies: Two - three fold increase in Supporting studies: Two - three fold increase in fertilization rates in mouse oocytes which were decreased by fertilization rates in mouse oocytes which were decreased by PAF antagonist WEB-2086 (Shi PAF antagonist WEB-2086 (Shi et alet al, 1992)., 1992).PAF concentrations increased with the advancing stages of PAF concentrations increased with the advancing stages of development of murine embryos thus suggesting its role in development of murine embryos thus suggesting its role in embryo cleavage (Ripps embryo cleavage (Ripps et alet al, 1993)., 1993).
Supporting studies: Significantly higher mitotic index of Supporting studies: Significantly higher mitotic index of mouse blastocysts with PAF.mouse blastocysts with PAF.Recent report- IVF rates were increased when PAF was Recent report- IVF rates were increased when PAF was incorporated in capacitation and fertilization medium in incorporated in capacitation and fertilization medium in murine embryos (Sarvanan and Sharma, 1997).murine embryos (Sarvanan and Sharma, 1997).
(iii) Implantation(iii) Implantation
Embryo derived PAF has been postulated to be the signal Embryo derived PAF has been postulated to be the signal required to induce EPF during the implantation stage of required to induce EPF during the implantation stage of pregnancy (Orozco pregnancy (Orozco et alet al, 1986)., 1986).
PAF antagonist viz. iloprost, SRI–63-441 is known to cause PAF antagonist viz. iloprost, SRI–63-441 is known to cause inhibition of implantation of mouse embryos which could inhibition 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 52021 Intra-uterine administration of PAF antagonist BN 52021 was known to inhibit the ovo-implantation in rats in a dose was known to inhibit the ovo-implantation in rats in a dose dependent fashion (Acker dependent fashion (Acker et alet al, 1988)., 1988).
Two cell mice embryos cultured in medium with PAF had Two 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 the Also enhanced the in vitroin vitro implantation rate in later stages implantation rate in later stages of embryo development (Nishi of embryo development (Nishi et alet al, 1995)., 1995).
(iv) Pregnancy and Parturition(iv) Pregnancy and ParturitionPAF is known to be produced by human embryos after IVF PAF is known to be produced by human embryos after IVF and embryo transfer – one of the earliest signals for the and 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 intra PAF may play a role in foetal lung maturation as after intra peritoneal administration into foetus, it caused decrease in peritoneal administration into foetus, it caused decrease in foetal pulmonary and hepatic glycogen concentration with foetal pulmonary and hepatic glycogen concentration with concomitant increase in pulmonary and hepatic plasma concomitant increase in pulmonary and hepatic plasma lactate levels.lactate levels.PAF present in the amniotic fluid of the pregnant women PAF present in the amniotic fluid of the pregnant women with a concentration increasing mid gestation onwards and with a concentration increasing mid gestation onwards and becoming very high just before the onset of labor, thus becoming very high just before the onset of labor, thus suggesting the role of PAF in parturition (Bernal suggesting the role of PAF in parturition (Bernal et alet al, 1991)., 1991).PAF receptor antagonist 1-659, 989 are known to prolong the PAF receptor antagonist 1-659, 989 are known to prolong the duration of parturition by 2-5 folds upon administration in duration of parturition by 2-5 folds upon administration in pregnant rats.pregnant rats.PAF receptor antagonist interfere in the normal progression PAF receptor antagonist interfere in the normal progression of events in parturition and PAF may be one of the mediaters of events in parturition and PAF may be one of the mediaters for the initiation of myometrical contractions necessary for for the initiation of myometrical contractions necessary for the expulsion of foetus (Yan-Pig Zhu the expulsion of foetus (Yan-Pig Zhu et alet al, 1991)., 1991).
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) was PAF acetyl hydrolase activity (which inactivates the PAF) was found to be present in human, bovine, hamster and rabbit found 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 human PAF increases the mean swimming speed of human spermatozoa at nano molar concentrations (Ricker spermatozoa at nano molar concentrations (Ricker et alet al, 1989)., 1989).
At micro molar concentration, it may decrease the motility and At 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; Ricker Ricker et alet al, 1989)., 1989).
Spermatozoa undergo AR without rapid loss of motility at Spermatozoa undergo AR without rapid loss of motility at micromolar concentration in cattlemicromolar concentration in cattle . .
Cattle Cattle : 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.
Possible mechanism of PAF in Capacitation and AR of Possible 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
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)
Mo
tilit
y (%
)
Control
50
100
150
200
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)
Mo
tilit
y (%
)
Control
50
100
150
200
Values based on 25 ejaculatesValues based on 25 ejaculates
Effect of PAF on Motility of Murrah spermatozoa (fresh)
01020304050607080
0 15 30 60 120
Duration of incubation (min)
Mo
tilit
y (%
)
Control
50
100
150
200
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)
Ac
ros
om
r R
ea
cti
on
(%
)
Control
50
100
150
200
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)
Ac
ros
om
e R
ea
cti
on
(%
)
Control
50
100
150
200
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)
Ac
ros
om
e R
ea
cti
on
(%
)
Control
50
100
150
200
Effect of 100 M PAF om Motility and Acrosome Reaction of Karan Fries spermatozoa (fresh)
0
20
40
60
80
0 15 30 60 120
Duration of incubation (min)
Perc
enta
ge V
alue
MOT
AR
Effect of 100 UM PAF on Motility and Acrosome Reaction of Karan Swiss spermatozoa (fresh)
0
20
40
60
80
0 15 30 60 120
Duration of incubation (min)
Parc
enta
ge V
alue
MOT
AR
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)
Pae
rcen
tage
Val
ue
MOT
AR
Relative changes in percent moti l i ty and acrosome reaction Relative changes in percent moti l i ty and acrosome reaction in Karan Swiss - fresh and frozen spermatozoa at the end of in Karan Swiss - fresh and frozen spermatozoa at the end of 15 and 30 min of incubation (at 100 15 and 30 min of incubation (at 100 µµ M of PAF).M of PAF).
CharacterCharacter Semen Semen TypeType
Time of ReactionTime of Reaction % change % change from 0 to from 0 to 15 min15 min
% change % change from 15 to from 15 to
30 min30 min0 min0 min 15 min15 min 30 min30 min
MotilityMotility(mean (mean ±± SE) SE)
FreshFresh 71.8 71.8 ±± 1.051.05
61.2 61.2 ±± 1.481.48
55.8 55.8 ±± 1.691.69
-14.76-14.76 -8.82-8.82
FrozenFrozen 51.3 51.3 ±± 1.911.91
45.7 45.7 ±± 1.551.55
41.7 41.7 ±± 1.741.74
-10.92-10.92 -8.75-8.75
ARAR(mean (mean ±± SE) SE)
FreshFresh 4.7 4.7 ±± 0.51 0.51 45.0 45.0 ±± 2.622.62
48.1 48.1 ±± 2.672.67
+857.45+857.45 +6.89+6.89
FrozenFrozen 18.9 18.9 ±± 2.792.79
33.4 33.4 ±± 3.603.60
34.6 34.6 ±± 3.433.43
+43.41+43.41 +3.59+3.59
Relative changes in percent moti l i ty and acrosome reaction Relative changes in percent moti l i ty and acrosome reaction in Karan Fries- fresh and frozen spermatozoa at the end of in Karan Fries- fresh and frozen spermatozoa at the end of 15 and 30 min of incubation (at 100 15 and 30 min of incubation (at 100 µµ M of PAF).M of PAF).
CharacterCharacter Semen Semen TypeType
Time of ReactionTime of Reaction % change % change from 0 to from 0 to 15 min15 min
% change % change from 15 to from 15 to
30 min30 min0 min0 min 15 min15 min 30 min30 min
MotilityMotility(mean (mean ±± SE) SE)
FreshFresh 72.6 72.6 ±± 0.790.79
62.9 62.9 ±± 1.121.12
58.6 58.6 ±± 1.181.18
-13.36-13.36 -6.84-6.84
FrozenFrozen 45.3 45.3 ±± 1.731.73
39.3 39.3 ±± 2.07 2.07
34.3 34.3 ±± 1.991.99
-13.25-13.25 -12.72-12.72
ARAR(mean (mean ±± SE) SE)
FreshFresh 3.6 3.6 ±± 0.25 0.25 45.6 45.6 ±± 1.751.75
46.4 46.4 ±± 1.051.05
+1166.67+1166.67 +1.75+1.75
FrozenFrozen 19.5 19.5 ±± 2.512.51
37.7 37.7 ±± 1.841.84
39.0 39.0 ±± 2.272.27
+93.33+93.33 +3.45+3.45
Relative changes in percent moti l i ty and acrosome reaction Relative changes in percent moti l i ty and acrosome reaction in Murrah- fresh and frozen spermatozoa at the end of 15 in Murrah- fresh and frozen spermatozoa at the end of 15 and 30 min of incubation (at 100 and 30 min of incubation (at 100 µµ M of PAF).M of PAF).
CharacterCharacter Semen Semen TypeType
Time of ReactionTime of Reaction % change % change from 0 to from 0 to 15 min15 min
% change % change from 15 to from 15 to
30 min30 min0 min0 min 15 min15 min 30 min30 min
MotilityMotility(mean (mean ±± SE) SE)
FreshFresh 72.6 72.6 ±± 1.241.24
60.0 60.0 ±± 1.671.67
54.8 54.8 ±± 1.461.46
-17.36-17.36 -8.67-8.67
FrozenFrozen 56.3 56.3 ±± 1.531.53
52.3 52.3 ±± 1.551.55
46.7 46.7 ±± 1.611.61
-7.11-7.11 -10.71-10.71
ARAR(mean (mean ±± SE) SE)
FreshFresh 4.4 4.4 ±±0.630.63 34.2 34.2 ±± 1.411.41
37.5 37.5 ±± 1.351.35
+677.27+677.27 +9.65+9.65
FrozenFrozen 17.5 17.5 ±± 2.842.84
27.3 27.3 ±± 1.841.84
28.9 28.9 ±± 2.052.05
+56.00+56.00 +5.66+5.66
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
HY
AL
UR
ON
IDA
SE
AC
TIV
ITY
(U
NIT
S)
CONTROLS
TREATED
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
HY
AL
UR
ON
IDA
SE A
CT
IVIT
Y (
UN
ITS
)
CONTROLS
TREATED
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
FER
TIL
IZA
TIO
N IN
DEX
CONTROLS
TREATED
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
FER
TIL
IZA
TIO
N IN
DEX
CONTROLS
TREATED
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
Rate of in vitro fert i l izat ion in dif ferent group of experiments.Rate of in vitro fert i l izat ion in dif ferent 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 from Effect 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 fertilization Effect 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 in Effect 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 %
A= Sperm control and no PAF treatment to IVF and IVCA= Sperm control and no PAF treatment to IVF and IVCB= Sperm control and PAF treatment to IVF and IVCB= Sperm control and PAF treatment to IVF and IVCC= Sperm treated and no PAF treatment to IVF and IVCC= Sperm treated and no PAF treatment to IVF and IVCD= 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
Dev
elop
men
t Rat
e (%
)
A
B
C
D
ConclusionConclusion
