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 />
150 Poster Abstracts<br />
transgenic constructs conta<strong>in</strong><strong>in</strong>g IR1 or IR2, each <strong>in</strong>verted repeat was<br />
transferred to pRNAi-ZP3 cassette (Ste<strong>in</strong> et al., 2003), to produce<br />
pZP3-oog1IR1 or pZP3-oog1IR2. In these transgenic constructs, the<br />
expression of Oog1 dsRNA hairp<strong>in</strong>s is controlled by ZP3 promoter<br />
which directs oocyte-specific expression. To analyze the phenotype of<br />
the transgenic females, each foun<strong>de</strong>r females (C57BL/6J) was mated<br />
with the same four wild type males (C57BL/6J) <strong>in</strong> a random<br />
sequence. Successful mat<strong>in</strong>g was verified by <strong>de</strong>tect<strong>in</strong>g vag<strong>in</strong>al plug.<br />
Mated females were then housed separately for observation and<br />
record<strong>in</strong>g the number of pups <strong>de</strong>livered per litter, which was averaged<br />
to assess fertility. To test the suppressive effect of dsRNAs, fertilized<br />
embryos were collected from transgenic F1 females mated with wildtype<br />
males 24 h after eCG <strong>in</strong>jection. Fertilized embryos were cultured<br />
<strong>in</strong> KSOM medium and their <strong>de</strong>velopment was recor<strong>de</strong>d at 24 h<br />
<strong>in</strong>terval until 96 h after eCG. At the time of embryo collection, GV<br />
stage oocytes were collected from the same female ovaries to exam<strong>in</strong>e<br />
the amount of Oog1 mRNA by real-time RT-PCR. We obta<strong>in</strong>ed n<strong>in</strong>e<br />
transgenic foun<strong>de</strong>rs and four female transgenic l<strong>in</strong>es out of 5 were<br />
sub-fertile. Embryos recovered from some transgenic F1 females<br />
mated with wild-type males exhibit <strong>de</strong>velopmental block <strong>in</strong> culture.<br />
These results suggest that Oog1 functions <strong>in</strong> early embryo<br />
<strong>de</strong>velopment <strong>in</strong> mouse. This approach provi<strong>de</strong>s a powerful method to<br />
study the function of the genes transcribed dur<strong>in</strong>g oogenesis and early<br />
embryogenesis.<br />
P372<br />
Clon<strong>in</strong>g and sequence analysis of Sheep fertil<strong>in</strong> β and its<br />
tissue expression<br />
Narenhua, N 1 *, Fu, L 1 , Li, N 1 , Bao, XRG 2<br />
1College of Animal Science and Medic<strong>in</strong>e, Inner Mongolia Agriculture<br />
University, Ch<strong>in</strong>a; 2 Life Science of College, Ch<strong>in</strong>a<br />
Fertil<strong>in</strong> β is member of the ADAMs (metalloprote<strong>in</strong>ase-like,<br />
dis<strong>in</strong>tegr<strong>in</strong>-like, cyste<strong>in</strong>e-rich) prote<strong>in</strong> family and is expressed on the<br />
sperm surface where it has been proposed to play a role <strong>in</strong> mammalian<br />
fertilization. Inhibition of the sperm-egg b<strong>in</strong>d<strong>in</strong>g and sperm-egg<br />
fusion make fertil<strong>in</strong> an attractive target for <strong>de</strong>velopment of an<br />
immunocontraceptive vacc<strong>in</strong>e. This study exam<strong>in</strong>ed fertil<strong>in</strong> β gene<br />
activity <strong>in</strong> relation to fertilization <strong>in</strong> the ram testis. Mixed primers for<br />
the polymerase cha<strong>in</strong> reaction (PCR) were <strong>de</strong>signed based on the high<br />
sequence homology of selected regions of known bov<strong>in</strong>e fertil<strong>in</strong> β<br />
gene. PCR-amplified cDNA fragments generated by 3\' and 5\' rapid<br />
amplification of cDNA ends (RACE) were comb<strong>in</strong>ed to generate fulllength<br />
cDNA sequence here for the first time. The 2217 bp cDNA has<br />
an open read<strong>in</strong>g frame encod<strong>in</strong>g 738 am<strong>in</strong>o acids, with a molecular<br />
mass of ~82 KDa. The <strong>de</strong>duced am<strong>in</strong>o acid sequence showed i<strong>de</strong>ntity<br />
at equivalent regions of bov<strong>in</strong>e (87.1 %), porc<strong>in</strong>e (73.2 %), mouse<br />
(37.6%), human (58.1%), and macaque (58.0%). Bov<strong>in</strong>e fertil<strong>in</strong> β<br />
conta<strong>in</strong>s a doma<strong>in</strong> with homology to dis<strong>in</strong>tegr<strong>in</strong>s, snake venom<br />
prote<strong>in</strong>s that b<strong>in</strong>d to <strong>in</strong>tegr<strong>in</strong>s via an <strong>in</strong>tegr<strong>in</strong>-b<strong>in</strong>d<strong>in</strong>g doma<strong>in</strong><br />
conta<strong>in</strong><strong>in</strong>g the tripepti<strong>de</strong> RGD. This partial <strong>in</strong>hibition of fusion with<br />
RGD pepti<strong>de</strong>s prompted the clon<strong>in</strong>g of the sheep homologue of<br />
bov<strong>in</strong>e fertil<strong>in</strong> β to <strong>de</strong>term<strong>in</strong>e if it possessed the tripepti<strong>de</strong> RGD or<br />
different am<strong>in</strong>o acid sequence <strong>in</strong> its dis<strong>in</strong>tegr<strong>in</strong> doma<strong>in</strong>. The<br />
dis<strong>in</strong>tegr<strong>in</strong> doma<strong>in</strong> of sheep has the tripepti<strong>de</strong> TDE (<strong>in</strong>stead of RGD)<br />
<strong>in</strong> its cell recognition region. In the present <strong>in</strong>vestigation we also<br />
report RT-PCR and <strong>in</strong> situ hybridization studies that show that the<br />
sheep fertil<strong>in</strong> β is transcripted only <strong>in</strong> adult ram testis and not <strong>in</strong> the<br />
all 3 epididymal regions. In situ transcript hybridization shows the<br />
transcript to be localized <strong>in</strong> round and enlongat<strong>in</strong>g spermatids <strong>in</strong> the<br />
sem<strong>in</strong>iferous epithelium. The work confirms that fertil<strong>in</strong> β is<br />
expressed tissue specifically. Key word: fertil<strong>in</strong> β/dis<strong>in</strong>tegr<strong>in</strong>/RGD<br />
pepti<strong>de</strong>/b<strong>in</strong>d<strong>in</strong>g and fusion<br />
P373<br />
Changes <strong>in</strong> ser<strong>in</strong>e/threon<strong>in</strong>e phosphorylation associated<br />
to the achievement of “<strong>in</strong> vitro” capacitation <strong>in</strong> boar<br />
spermatozoa<br />
Ramió-Lluch, L* and Rodriguez-Gil, JE<br />
Unit of animal <strong>Reproduction</strong>, Dept. Animal Medic<strong>in</strong>e and Surgery, School of<br />
Veter<strong>in</strong>ary Medic<strong>in</strong>e, Autonomous University of Barcelona, Bellaterra, Spa<strong>in</strong><br />
Several studies <strong>in</strong> different species have shown that tyros<strong>in</strong>e<br />
phosphorylation of sperm prote<strong>in</strong>s is a suitable marker of the<br />
capacitation. Furthermore, capacitation has been l<strong>in</strong>ked to changes <strong>in</strong><br />
the spatial location of tyros<strong>in</strong>e-phosphorylation. However, there is<br />
little <strong>in</strong>formation about changes on both ser<strong>in</strong>e- and threon<strong>in</strong>ephosphorylation<br />
status dur<strong>in</strong>g sperm capacitation. Thus, the ma<strong>in</strong> aim<br />
of this study has was the observation of putative changes <strong>in</strong> both<br />
expression and location of ser<strong>in</strong>e- and threon<strong>in</strong>e phosphorylation<br />
dur<strong>in</strong>g "<strong>in</strong> vitro" capacitation (IVC) of boar sperm. For this purpose,<br />
boar sperm from fresh ejaculates were <strong>in</strong>cubated <strong>in</strong> specific<br />
capacitation medium dur<strong>in</strong> 4 hours at 39ºC <strong>in</strong> a 5%CO2 atmosphere .<br />
Sperm aliquots were taken at 0, 1, 2 , 3 and 4 hours after <strong>in</strong>cubation<br />
and both the general presence and spatial location of ser<strong>in</strong>e- and<br />
threon<strong>in</strong>e-phosphorylation were evaluated.<br />
Our results showed that boar sperm had an specific pattern of<br />
phosphorylation <strong>in</strong> both ser<strong>in</strong>e and threon<strong>in</strong>e residues. Weight of the<br />
ma<strong>in</strong> bands were around 25-30 KDa, 30-35 KDa and 50 KDa <strong>in</strong><br />
ser<strong>in</strong>e-phosphorylation, and around 30 KDa, 50 KDa and 75 KDa <strong>in</strong><br />
threon<strong>in</strong>e-phosphorylation. IVC <strong>in</strong>duced an observable rise <strong>in</strong> the<br />
expression of ser<strong>in</strong>e phosphorilation dur<strong>in</strong>g capacitation, specially <strong>in</strong><br />
the band around 30-35 KDa <strong>in</strong> both cases. These results were<br />
accompanied with specific changes <strong>in</strong> the spatial location of both<br />
ser<strong>in</strong>e- and threon<strong>in</strong>e-phosphorylation <strong>in</strong> spermatozoa. All these<br />
results <strong>in</strong>dicate that IVC is associated with specific changes <strong>in</strong> prote<strong>in</strong><br />
phosphorylation, <strong>in</strong> tyros<strong>in</strong>e residues and <strong>in</strong> both ser<strong>in</strong>e- and<br />
threon<strong>in</strong>e residues as well. F<strong>in</strong>ally, the observed spatial changes <strong>in</strong><br />
prote<strong>in</strong> phosphorylation <strong>in</strong>dicates that they could be <strong>in</strong>volved <strong>in</strong> the<br />
achievement of a feasible IVC.<br />
P374<br />
Sperm DNA fragmentation and presence of vary<strong>in</strong>g levels<br />
of protam<strong>in</strong>e 1 mRNA <strong>in</strong> Ram and Goat<br />
Roy, R 1 *, Gosalbez, A 1 , Lopez-Fernan<strong>de</strong>z, C 1 , Arroyo, F 1 , García-Hurtado, J 1 ,<br />
Casado, S 2 , De La Torre, J 1 , Gosalvez, J 1<br />
1Biology, Universidad Autonoma <strong>de</strong> Madrid, Spa<strong>in</strong>; 2 Research And<br />
Development, Halotech Sl, Spa<strong>in</strong><br />
Sperm DNA fragmentation has been the subject of numerous studies<br />
because the <strong>in</strong>ci<strong>de</strong>nce of a high rate of nuclei conta<strong>in</strong><strong>in</strong>g damaged<br />
DNA is highly correlated with a loss of fertility. However, the orig<strong>in</strong><br />
of DNA fragmentation is mostly unknown, although apoptosis,<br />
oxidative stress, or persistence of DNA breaks produced dur<strong>in</strong>g<br />
chromat<strong>in</strong> protam<strong>in</strong>ation <strong>in</strong> spermiogenesis, could be direct causes of<br />
chromat<strong>in</strong> damage. The aim of the present <strong>in</strong>vestigation was to<br />
analyze the correlation between different levels of protam<strong>in</strong>e 1 mRNA<br />
<strong>in</strong> sperm cells from ejaculated semen samples <strong>in</strong> ram and goat with<br />
the dynamics of DNA fragmentation after ejaculation and sperm<br />
extension. For this purpose, 10 rams from Castellana breed and 10<br />
goats all show<strong>in</strong>g Sperm DNA Fragmentation (SDF) <strong>in</strong><strong>de</strong>x below 6%<br />
were assessed for SDF by us<strong>in</strong>g the Sperm Chromat<strong>in</strong> Dispersion test<br />
(SCD; Halomax). The dynamics of SDF was assessed at T0,T1,T4<br />
and T24 (numbers <strong>in</strong>dicates hours after ejaculation <strong>in</strong> sperm samples<br />
exten<strong>de</strong>d and <strong>in</strong>cubated at 37ºC). SDF Values were plotted aga<strong>in</strong>st<br />
<strong>in</strong>creas<strong>in</strong>g <strong>in</strong>cubation times. Results showed that differences <strong>in</strong> the<br />
dynamic range of SDF do exist among different animals; each animal<br />
reached a SDF <strong>in</strong><strong>de</strong>x 50% of DNA fragmentation at different times<br />
(rang<strong>in</strong>g from 4 to 24 h). Simultaneously, the amount of protam<strong>in</strong>e 1<br />
mRNA from mature spermatozoa was analyzed us<strong>in</strong>g real-time PCR.<br />
Interest<strong>in</strong>gly, when levels of protam<strong>in</strong>e 1 mRNA was compared with<br />
the dynamic range of SDF present <strong>in</strong> the same samples, those<br />
<strong>in</strong>dividuals show<strong>in</strong>g a rapid <strong>in</strong>crease <strong>in</strong> sperm DNA fragmentation,<br />
exhibited the lower levels of protam<strong>in</strong>e 1 mRNA. These results<br />
suggest that sperm chromat<strong>in</strong> fail<strong>in</strong>g to achieve a proper