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 />
Poster Abstracts 149<br />
<strong>in</strong> the composition of the mucous layer of the endometrium/cervix of<br />
mares with Persistent Post-Breed<strong>in</strong>g Endometritis<br />
(PPBEM)/Ascend<strong>in</strong>g Placentitis (AP), which affect normal barrier<br />
function at these sites. Both of these conditions cause consi<strong>de</strong>rable<br />
economic losses to the Irish equ<strong>in</strong>e bloodstock <strong>in</strong>dustry. PPBEM<br />
manifests as a failure to clear semen, exogenous contam<strong>in</strong>ants and<br />
bacteria from the endometrium after breed<strong>in</strong>g, lead<strong>in</strong>g to an embryotoxic<br />
environment and failure to conceive. In AP, the cervical mucus<br />
plug is breached by <strong>in</strong>vad<strong>in</strong>g microorganisms late <strong>in</strong> pregnancy,<br />
<strong>in</strong>duc<strong>in</strong>g abortion or premature birth. In an attempt to address this<br />
hypothesis, our <strong>in</strong>itial objective has been to <strong>de</strong>term<strong>in</strong>e the expression<br />
of muc<strong>in</strong> genes with<strong>in</strong> the reproductive tract of the mare. The<br />
expression of the muc<strong>in</strong> prote<strong>in</strong>s is enco<strong>de</strong>d by approximately 20<br />
genes and is un<strong>de</strong>r the <strong>in</strong>fluence of steroid hormones dur<strong>in</strong>g the<br />
oestrus cycle. We surveyed 16 muc<strong>in</strong> genes of the normal equ<strong>in</strong>e<br />
vag<strong>in</strong>a, cervix, endometrium and uter<strong>in</strong>e tube dur<strong>in</strong>g oestrus (n=6)<br />
and dioestrus (n=12). Orthologues of human muc<strong>in</strong> genes were<br />
i<strong>de</strong>ntified <strong>in</strong> the horse genome and their expression <strong>de</strong>term<strong>in</strong>ed by<br />
PCR and agarose gel-electrophoresis. MUC1, a membrane-bound<br />
muc<strong>in</strong>, and MUC2, a secreted muc<strong>in</strong>, are expressed <strong>in</strong> the human and<br />
equ<strong>in</strong>e reproductive tract. MUC3 appears to be expressed <strong>in</strong> the<br />
equ<strong>in</strong>e endometrium only dur<strong>in</strong>g dioestrus, while MUC5B is<br />
expressed only dur<strong>in</strong>g oestrus. MUC5AC is one of the secreted<br />
muc<strong>in</strong>s that are expressed <strong>in</strong> very low concentrations <strong>in</strong> the human<br />
reproductive tract and it is expressed <strong>in</strong> the equ<strong>in</strong>e lung. However, it<br />
is not expressed <strong>in</strong> the equ<strong>in</strong>e reproductive tract. MUC7, which is not<br />
expressed <strong>in</strong> the human reproductive tract, is expressed <strong>in</strong> the mare.<br />
This study provi<strong>de</strong>s basel<strong>in</strong>e data for normal mares and for future<br />
comparison with mares affected by PPBEM or AP. Future work will<br />
<strong>in</strong>clu<strong>de</strong> real time PCR and <strong>in</strong>- situ hybridisation to <strong>de</strong>term<strong>in</strong>e gene<br />
expression levels and tissue distribution with<strong>in</strong> the reproductive tract<br />
throughout the oestrus cycle <strong>in</strong> healthy and diseased mares.<br />
P369<br />
Endometrial expression of IGF-I, IGF-II and IGF-1R<br />
throughout the cow oestrous cycle<br />
Meikle, A 1 *, Carriquiry, M 2 , Chalar, C 3 , Sangu<strong>in</strong>etti, C 3 , Abreu, C 3 , Crespi, D 1 ,<br />
Cavestany, D 1<br />
1Laboratory of Nuclear Techniques, Veter<strong>in</strong>ary Faculty of Uruguay,<br />
Uruguay; 2 Agronomy Faculty, Uruguay; 3 Sciences Faculty, Uruguay<br />
The <strong>in</strong>sul<strong>in</strong>-like growth factor (IGF) system is expressed <strong>in</strong> bov<strong>in</strong>e<br />
uterus dur<strong>in</strong>g the estrous cycle and early pregnancy and plays an<br />
important role <strong>in</strong> regulat<strong>in</strong>g the <strong>de</strong>velopment of the embryo and<br />
uterus. IGF-I and -II mediate their effects through the type 1 IGF<br />
receptor (IGF-1R). In this study, the expression of the IGFs and IGF-<br />
1R was <strong>de</strong>term<strong>in</strong>ed on endometrial transcervical biopsies collected on<br />
days 0 (oestrus), 5, 12 and 19 of the cow oestrous cycle (n = 8). The<br />
abundance of mRNA of IGF-I, IGF-II, IGF-1R, and an endogenous<br />
control (ribosomal prote<strong>in</strong> L19; RPL19) was measured by quantitative<br />
realtime RT-PCR us<strong>in</strong>g SYBR Green. Abundance of mRNA of target<br />
genes was normalized to RPL19 and expressed <strong>in</strong> relative amounts to<br />
an external control (ΔΔCT -method). Results were analyzed with a<br />
repeated measures analysis (PROC MIXED of SAS) and consi<strong>de</strong>red<br />
to differ when P < 0.05. The expression of RPL19 mRNA did not<br />
throughout the oestrus cycle. Endometrial expression of IGF-I mRNA<br />
was the greatest at oestrus and day 5 (100%), and <strong>de</strong>creased to 47%<br />
and 35% of the <strong>in</strong>itial values on days 12 and 17, respectively.<br />
Abundance of IGF-II mRNA peaked at day 12 and <strong>de</strong>creased sharply<br />
thereafter (to one third of day 12 values). Interest<strong>in</strong>gly, IGF-1R<br />
mRNA expression showed the same pattern dur<strong>in</strong>g the oestrous cycle<br />
that IGF-II mRNA. IGF-1R mRNA showed reduced expression at<br />
oestrus and day 5, <strong>in</strong>creased by 1-fold on day 12, and <strong>de</strong>creased on<br />
day 17 to values similar to the oestrus ones. These results show that<br />
these IGF system components are dist<strong>in</strong>ctively regulated dur<strong>in</strong>g the<br />
oestrous cycle suggest<strong>in</strong>g that modulation of IGF system may<br />
<strong>in</strong>fluence uter<strong>in</strong>e activity dur<strong>in</strong>g this period. The earlier and later<br />
<strong>in</strong>creases found on IGF-I and IGF-II respectively, suggest a<br />
differential role of these hormones on the early/late blastocyst and/or<br />
on the regulation of uter<strong>in</strong>e function. The <strong>in</strong>crease <strong>in</strong> the uter<strong>in</strong>e<br />
sensitivity to IGFs dur<strong>in</strong>g the late luteal phase – by IGF-1R<br />
expression- may re<strong>in</strong>force the role of IGF-II dur<strong>in</strong>g the early<br />
pregnancy.<br />
P370<br />
Regulation of Lefty2 <strong>in</strong> the oviduct of cyclic, pregnant,<br />
and pseudopregnant rats.<br />
Argañaraz, ME 1 , Val<strong>de</strong>cantos, PA 2 , Miceli, DC 1 *<br />
1Instituto Superior <strong>de</strong> Investigaciones Biológicas, CONICET, Argent<strong>in</strong>a;<br />
2<strong>Facultad</strong> <strong>de</strong> Bioquímica,Cátedra <strong>de</strong> Biología Celular, Universidad Nacional<br />
<strong>de</strong> Tucumán, Argent<strong>in</strong>a<br />
Transform<strong>in</strong>g growth factor beta superfamily members are closely<br />
associated with tissue remo<strong>de</strong>l<strong>in</strong>g events and reproductive processes,<br />
be<strong>in</strong>g <strong>in</strong>volved <strong>in</strong> the embryo <strong>de</strong>velopment and <strong>in</strong> the maternalembryo<br />
cross-talk. Moreover, transform<strong>in</strong>g growth factor beta and<br />
their receptors were found <strong>in</strong> the preimplantation embryo and the<br />
reproductive tract (oviduct and uterus). Lefty2 an unusual member of<br />
this family has been implicated <strong>in</strong> the regulation of other transform<strong>in</strong>g<br />
growth factor beta members such as nodal, activ<strong>in</strong>, bone morphogenic<br />
prote<strong>in</strong>s and transform<strong>in</strong>g growth factor beta 1 via cryto co-receptors<br />
and by an antagonic mechanism. To date, the presence and regulation<br />
of Lefty2 <strong>in</strong> the rat oviduct have not been <strong>de</strong>scribed yet. The aim of<br />
the present study was to <strong>in</strong>vestigate the expression of Lefty2 and its<br />
co-receptor (crypto) <strong>in</strong> the oviduct. RNA; oviductal prote<strong>in</strong>s and<br />
tissues were collected from cyclic non-pregnant, pregnant, and<br />
hormonally-<strong>in</strong>duced pseudopregnant rats. Lefty is a pre-proprote<strong>in</strong> of<br />
42 kDa that is proteolytic activated to a mature form of 26 kDa.<br />
Lefty2 prote<strong>in</strong>s were <strong>de</strong>tected by western blots <strong>in</strong> the oviduct of<br />
cyclic, pregnant, and pseudopregnant rats but were not <strong>in</strong>fluenced by<br />
the estrous cycle. Dur<strong>in</strong>g early pregnancy, Lefty2 mature form was<br />
significantly higher at day 4 (when the embryo is still <strong>in</strong> the oviduct)<br />
and then it was gradually <strong>de</strong>creased while the pre-proprote<strong>in</strong> had not<br />
significant modifications. Dur<strong>in</strong>g pseudopregnancy, both form of<br />
Lefty2 prote<strong>in</strong> were found at very low levels, without variations.<br />
Crypto transcripts, analyzed by semi-quantitative RT-PCR, were<br />
<strong>de</strong>tected <strong>in</strong> the oviduct <strong>in</strong> the three studied conditions. Crypto<br />
expression levels were <strong>in</strong>creased at day 4 of pregnancy, as we have<br />
reported for the lefty2 prote<strong>in</strong>. Neither the estrous cycle nor the<br />
pseudopregnancy showed variation <strong>in</strong> the crypto gene expression<br />
pattern. These results suggest that Lefty2 and crypto are present <strong>in</strong> the<br />
rat oviduct dur<strong>in</strong>g pregnancy; that Lefty2 could act along a paracr<strong>in</strong>e<br />
pathway by b<strong>in</strong>d<strong>in</strong>g to specific receptors on oviductal cells and that<br />
their expression could be <strong>in</strong><strong>de</strong>pen<strong>de</strong>nt of steroid regulation. The<br />
secretion of Lefty2 could be important for the embryo ma<strong>in</strong>tenance<br />
dur<strong>in</strong>g its pass through the oviduct.<br />
P371<br />
Oog1 is a maternal effect gene required for the<br />
<strong>de</strong>velopment of mouse preimplantation embryo<br />
M<strong>in</strong>ami, N 1 *, Imaichi, H 1 , Tsukamoto, S 2 , Ohta, Y 3 , Kito, S 3 , Kimura, K 4 , Imai, H<br />
1Lab. Reproductive Biology, Kyoto University, Japan; 2 Physiology and Cell<br />
Biology, Tokyo Medical and Dental University, Japan; 3 Research Center for<br />
Radiation Safety, National Institute of Radiological Sciences, Japan; 4 Animal<br />
Breed<strong>in</strong>g and <strong>Reproduction</strong> Research Team, National Institute of Livestock<br />
and Grassland Sci, Japan<br />
Previously we i<strong>de</strong>ntified an oocyte-specific gene, Oog1 (M<strong>in</strong>ami et<br />
al., 2001). The expression of Oog1 starts at E15.5 <strong>in</strong> embryonic ovary<br />
and cont<strong>in</strong>ues until the 2-cell stage. The expression is dramatically<br />
<strong>de</strong>gra<strong>de</strong>d thereafter. The most <strong>in</strong>terest<strong>in</strong>g characteristic of Oog1<br />
prote<strong>in</strong> is nuclear accumulation dur<strong>in</strong>g the late 1-cell to early 2-cell<br />
stage (M<strong>in</strong>ami et al., 2003). The period co<strong>in</strong>ci<strong>de</strong>s with the time of<br />
zygotic gene activation and the time of first mitosis. The molecular<br />
biological feature of Oog1 is the association with small GTP b<strong>in</strong>d<strong>in</strong>g<br />
prote<strong>in</strong>s, such as Ras and Ran (Tsukamoto et al., 2006). However, the<br />
precise mechanism of Oog1 <strong>in</strong> embryonic <strong>de</strong>velopment rema<strong>in</strong>s<br />
unknown. In addition, s<strong>in</strong>ce Oog1 is multicopy gene, knockout<br />
approach can not be used. In the present study, we exam<strong>in</strong>ed the role<br />
of Oog1 <strong>in</strong> the <strong>de</strong>velopment of mouse embryos us<strong>in</strong>g transgenic<br />
RNAi approach. Two constructs differ<strong>in</strong>g <strong>in</strong> the sequence of Oog1<br />
<strong>in</strong>verted repeats (IR1 and IR2) were employed <strong>in</strong> this study. To make