Reproduction in Domestic Animals - Facultad de Ciencias Veterinarias
Reproduction in Domestic Animals - Facultad de Ciencias Veterinarias
Reproduction in Domestic Animals - Facultad de Ciencias Veterinarias
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16 t h International Congress on Animal <strong>Reproduction</strong><br />
148 Poster Abstracts<br />
were observed <strong>in</strong> the sem<strong>in</strong>iferous tubules. The proliferation of germ<br />
cells resumed and the differentiated spermatogonia appeared aga<strong>in</strong> at<br />
6 weeks. The spermatogenesis was found to restore completely by 11<br />
weeks after the <strong>in</strong>jection. Among the prote<strong>in</strong>s that reappeared<br />
concomitantly with the spermatids, we focused on a prote<strong>in</strong> migrated<br />
to pI 4.0 and 66kDa on 2-D-gel. It was i<strong>de</strong>ntified as a hypothetical<br />
prote<strong>in</strong>, NYD-SP26, by TOF-MS analysis. NYD-SP26 mRNA was<br />
found to be specifically expressed <strong>in</strong> elongate spermatids at steps 10-<br />
16 and its translates were first <strong>de</strong>tected at step 13 and ma<strong>in</strong>ly localized<br />
to the flagellar pr<strong>in</strong>cipal piece of the sperm. Furthermore it was found<br />
that NYD-SP26 had calcium-b<strong>in</strong>d<strong>in</strong>g properties.<br />
Conclusions NYD-SP26 is a new member of the calcium-b<strong>in</strong>d<strong>in</strong>g<br />
prote<strong>in</strong>s expressed <strong>in</strong> the elongate spermatids, which is subsequently<br />
localized <strong>in</strong>to the pr<strong>in</strong>cipal piece of flagella of matured sperm. These<br />
data <strong>in</strong>dicate that this prote<strong>in</strong> plays a part of [Ca 2+ ] i signall<strong>in</strong>g <strong>in</strong><br />
sperm.<br />
P366<br />
I<strong>de</strong>ntification of a novel ov<strong>in</strong>e acrosome prote<strong>in</strong>:<br />
implications for sex-sorted spermatozoa<br />
Leahy, T 1 *; Marti, JI 2 ; Evans, G 1 ; Maxwell, WMC 1<br />
1REPROGEN, Faculty of Veter<strong>in</strong>ary Science, The University of Sydney,<br />
Australia; 2 CITA, Aragon, Spa<strong>in</strong><br />
Sex-sort<strong>in</strong>g subjects spermatozoa to numerous stressors. Sorted<br />
spermatozoa are highly diluted and exposed to mechanical forces,<br />
<strong>in</strong>clud<strong>in</strong>g propulsion <strong>in</strong>to a collection tube at 90km/hr. This may<br />
<strong>de</strong>stabilise the sperm membrane by stripp<strong>in</strong>g prote<strong>in</strong>s from its surface.<br />
However, sorted ram spermatozoa have superior fertility to non-sorted<br />
spermatozoa 1 as the sort<strong>in</strong>g process gates out non-viable spermatozoa,<br />
leav<strong>in</strong>g a homogenous and possibly membrane-<strong>in</strong>tact population. The<br />
aim of this experiment was to <strong>de</strong>tect differences <strong>in</strong> the membrane<br />
prote<strong>in</strong> profiles of a) viable sorted spermatozoa b) non-viable sorted<br />
spermatozoa and c) non-sorted spermatozoa (control) and relate these<br />
to variations <strong>in</strong> sperm function.<br />
Semen was collected by artificial vag<strong>in</strong>a (n= 3 rams), diluted <strong>in</strong> a<br />
Tris-citrate-fructose buffer supplemented with 1% PVA and <strong>in</strong>cubated<br />
(1hr, 34°C) with 311uM Hoechst 33342. The <strong>in</strong>cubated sample was<br />
sorted to obta<strong>in</strong> a viable, non-viable and non-sorted (control)<br />
population. The sperm samples were centrifuged (7500g, 5 m<strong>in</strong>) to<br />
obta<strong>in</strong> a pellet that was resuspen<strong>de</strong>d <strong>in</strong> membrane prote<strong>in</strong> extraction<br />
medium (2% SDS, 28% sucrose, 12.4mM TEMED, 185mM Tris<br />
chlori<strong>de</strong>) and <strong>in</strong>cubated (100°C, 5 m<strong>in</strong>). The <strong>in</strong>cubated sample was<br />
centrifuged (7500g, 5 m<strong>in</strong>) and the prote<strong>in</strong>s <strong>in</strong> the supernatant<br />
analysed us<strong>in</strong>g one- and two-dimensional gel electrophoresis and<br />
mass spectrometry.<br />
A prote<strong>in</strong> of 15kDa and isoelectric po<strong>in</strong>t of 4.99 was found <strong>in</strong><br />
abundance on the non-viable and non-sorted sperm membranes but<br />
was not present on the membranes of viable spermatozoa. This prote<strong>in</strong><br />
was i<strong>de</strong>ntified as an <strong>in</strong>tra-acrosomal, non-bacteriolytic, C lysozymelike<br />
prote<strong>in</strong>, termed SLLP1. This prote<strong>in</strong> is exposed dur<strong>in</strong>g the<br />
acrosome reaction, and reta<strong>in</strong>ed on the equatorial segment of the<br />
sperm membrane. The absence of this prote<strong>in</strong> <strong>in</strong> the viable, sorted<br />
sperm population suggests that sex-sort<strong>in</strong>g actively selects sperm that<br />
are acrosome-<strong>in</strong>tact. These results provi<strong>de</strong> evi<strong>de</strong>nce that the sort<strong>in</strong>g<br />
process selects a superior, homogenous population of membrane<strong>in</strong>tact<br />
spermatozoa. This may expla<strong>in</strong> why sorted spermatozoa survive<br />
longer <strong>in</strong> the female tract than non-sorted spermatozoa and can<br />
provi<strong>de</strong> acceptable fertility rates with low-dose <strong>in</strong>sem<strong>in</strong>ations 1 . This<br />
work was supported by XY Inc and the Major National Research<br />
Facilities Program (Biomedical No<strong>de</strong> of the Australian Proteome<br />
Analysis Facility). 1 <strong>de</strong> Graaf, S, et al. (2007) Reprod Dom Anim 42:<br />
648-653<br />
P367<br />
The effect of VEGF on the change of P44/p42 MAP<br />
k<strong>in</strong>ases, Prote<strong>in</strong> K<strong>in</strong>ase C and Prote<strong>in</strong> tyros<strong>in</strong>e k<strong>in</strong>ases of<br />
ov<strong>in</strong>e oocytes matured <strong>in</strong> vitro<br />
Hail<strong>in</strong>g, LUO 1 *, X<strong>in</strong>, CAO 1 ,2, P<strong>in</strong>g, ZHOU 3 , Youzhang, ZHAO 2 , Guoq<strong>in</strong>g,<br />
SHI 3<br />
1College of Animal Science and Technology, Ch<strong>in</strong>a Agriculture University,<br />
Beij<strong>in</strong>g 100094,P.R Ch<strong>in</strong>a; 2 College of Animal Science and Technology,<br />
Gansu Agricultural University, Lanzhou 730070, P.R Ch<strong>in</strong>a; 3 X<strong>in</strong>jiang<br />
Aca<strong>de</strong>mic of Agriculture and Reclamation Science, Shihezi, X<strong>in</strong>jiang, 832000,<br />
P.R Ch<strong>in</strong>a<br />
P44/p42 MAP k<strong>in</strong>ases (Erk1 and Erk2) and Prote<strong>in</strong> K<strong>in</strong>ase C (PKC)<br />
are mediate essential cellular signals required for activation,<br />
proliferation, differentiation and survival. Prote<strong>in</strong> Tyros<strong>in</strong>e K<strong>in</strong>ases<br />
(PTKs) perform a critical role <strong>in</strong> signal transduction pathways that<br />
control cell proliferation, differentiation, metabolism and apoptosis.<br />
Our previous results proved that Vascular Endothelial Growth Factor<br />
(VEGF) is a powerful mediator for vessel permeability and could<br />
improve the quality of ov<strong>in</strong>e oocyte maturation <strong>in</strong> vitro. In this study,<br />
to <strong>in</strong>vestigate the effect of VEGF on the change of phosphorylation<br />
ERKs, PKC and PTKs activity <strong>in</strong> ov<strong>in</strong>e oocyte, 5 ng/ml VEGF was<br />
supplied <strong>in</strong> media dur<strong>in</strong>g maturation <strong>in</strong> vitro and the method of<br />
ELISA was used to <strong>de</strong>term<strong>in</strong>e the ERKs, PKC and PTKs activities.<br />
The results shown that the levels of phosphorylation ERKs, PKC and<br />
PTKs activity <strong>in</strong> VEGF groups were higher than the without-VEGF<br />
groups. Moreover VEGF was strongly enhanced the level of<br />
phosphorylation ERKs and PKC activity but reduced PTKs activity<br />
dur<strong>in</strong>g the period of ov<strong>in</strong>e oocyte maturation <strong>in</strong> vitro. The<br />
phosphorylation levels of ERKs and PKC were slowly reduced from 0<br />
hour to germ<strong>in</strong>al vesicle breakdown (GVBD), subsequently<br />
phosphorylation ERKs level rapidly rose when GVBD was<br />
commenc<strong>in</strong>g and kept this higher level to MII phase, specially arrived<br />
at top at 18 hours and 21 hours. Phosphorylation PKC level took on<br />
fluctuant change but rose little by little and ma<strong>in</strong>ta<strong>in</strong>ed high level at<br />
MII phase, reached peak at 21 hours. The ERKs and PKC activity<br />
reached the highest at 21 hour <strong>in</strong> vitro culture and <strong>de</strong>scen<strong>de</strong>d aga<strong>in</strong><br />
from 21 hours to 24 hours but <strong>in</strong>terest<strong>in</strong>gly that the levels were mount<br />
up aga<strong>in</strong> <strong>in</strong> the VEGF group at 24 hours compared to the control. The<br />
level of PTKs activity rapidly lessened from 0 hour to GVBD,<br />
fluctuant changed and kept low level dur<strong>in</strong>g the residual period of<br />
oocyte maturation, but reached high level at 21 hours. Prote<strong>in</strong> tyros<strong>in</strong>e<br />
k<strong>in</strong>ase activity is often associated with membrane receptor prote<strong>in</strong><br />
tyros<strong>in</strong>e k<strong>in</strong>ases such as VEGFR. Phosphorylation of ERKs and PKC<br />
by PTKs is essential for the regulation of oocyte maturation biological<br />
mechanisms. It could hypothesis that activation of VEGFR-mediated<br />
pathways occurs by supply<strong>in</strong>g VEGF <strong>in</strong> ov<strong>in</strong>e oocyte maturation<br />
medium, which cross-l<strong>in</strong>k the VEGFR on the plasma membrane and<br />
<strong>in</strong>itiate receptor-mediated signal<strong>in</strong>g pathways, lead<strong>in</strong>g to ERKs and<br />
PKC activation by PTKs activity change. In conclusion, VEGF <strong>in</strong>duce<br />
the activation of ERK and PKC functions <strong>de</strong>pen<strong>de</strong>ntly of the<br />
activation of PTKs, strongly support<strong>in</strong>g the view that VEGF could<br />
enhance the ability of oocyte maturation.<br />
Keywords VEGF, p44/p42 MAP k<strong>in</strong>ases, Prote<strong>in</strong> K<strong>in</strong>ase C, Prote<strong>in</strong><br />
Tyros<strong>in</strong>e k<strong>in</strong>ases, ov<strong>in</strong>e oocyte<br />
The present study was supported by National Natural Science<br />
Foundation of Ch<strong>in</strong>a (No. 30371035)<br />
P368<br />
Muc<strong>in</strong> Gene Expression <strong>in</strong> the Equ<strong>in</strong>e Reproductive Tract<br />
Maischberger, E 1 *, Irw<strong>in</strong>, J 1 , Cumm<strong>in</strong>s, C 1 , Duggan, V 1 , Carr<strong>in</strong>gton, S 1 ,<br />
Corfield, A 2 , Reid, C 1<br />
1Veter<strong>in</strong>ary Sciences Centre, University College Dubl<strong>in</strong>- School of Agriculture,<br />
Food Science and Veter<strong>in</strong>ary Medic<strong>in</strong>e, Ireland; 2 Muc<strong>in</strong> research group,<br />
University of Bristol, United K<strong>in</strong>gdom<br />
Muc<strong>in</strong>s, which are the pr<strong>in</strong>cipal gel-form<strong>in</strong>g components of the mucus<br />
gel, play an important role <strong>in</strong> lubricat<strong>in</strong>g, hydrat<strong>in</strong>g and protect<strong>in</strong>g<br />
mucosal surfaces. In particular, they contribute to a barrier aga<strong>in</strong>st<br />
microbial <strong>in</strong>fection, tox<strong>in</strong>s and other potentially harmful elements <strong>in</strong><br />
the supramucosal environment. We hypothesize that there are changes