Reproduction in Domestic Animals - Facultad de Ciencias Veterinarias

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16 t h International Congress on Animal Reproduction 148 Poster Abstracts were observed in the seminiferous tubules. The proliferation of germ cells resumed and the differentiated spermatogonia appeared again at 6 weeks. The spermatogenesis was found to restore completely by 11 weeks after the injection. Among the proteins that reappeared concomitantly with the spermatids, we focused on a protein migrated to pI 4.0 and 66kDa on 2-D-gel. It was identified as a hypothetical protein, NYD-SP26, by TOF-MS analysis. NYD-SP26 mRNA was found to be specifically expressed in elongate spermatids at steps 10- 16 and its translates were first detected at step 13 and mainly localized to the flagellar principal piece of the sperm. Furthermore it was found that NYD-SP26 had calcium-binding properties. Conclusions NYD-SP26 is a new member of the calcium-binding proteins expressed in the elongate spermatids, which is subsequently localized into the principal piece of flagella of matured sperm. These data indicate that this protein plays a part of [Ca 2+ ] i signalling in sperm. P366 Identification of a novel ovine acrosome protein: implications for sex-sorted spermatozoa Leahy, T 1 *; Marti, JI 2 ; Evans, G 1 ; Maxwell, WMC 1 1REPROGEN, Faculty of Veterinary Science, The University of Sydney, Australia; 2 CITA, Aragon, Spain Sex-sorting subjects spermatozoa to numerous stressors. Sorted spermatozoa are highly diluted and exposed to mechanical forces, including propulsion into a collection tube at 90km/hr. This may destabilise the sperm membrane by stripping proteins from its surface. However, sorted ram spermatozoa have superior fertility to non-sorted spermatozoa 1 as the sorting process gates out non-viable spermatozoa, leaving a homogenous and possibly membrane-intact population. The aim of this experiment was to detect differences in the membrane protein profiles of a) viable sorted spermatozoa b) non-viable sorted spermatozoa and c) non-sorted spermatozoa (control) and relate these to variations in sperm function. Semen was collected by artificial vagina (n= 3 rams), diluted in a Tris-citrate-fructose buffer supplemented with 1% PVA and incubated (1hr, 34°C) with 311uM Hoechst 33342. The incubated sample was sorted to obtain a viable, non-viable and non-sorted (control) population. The sperm samples were centrifuged (7500g, 5 min) to obtain a pellet that was resuspended in membrane protein extraction medium (2% SDS, 28% sucrose, 12.4mM TEMED, 185mM Tris chloride) and incubated (100°C, 5 min). The incubated sample was centrifuged (7500g, 5 min) and the proteins in the supernatant analysed using one- and two-dimensional gel electrophoresis and mass spectrometry. A protein of 15kDa and isoelectric point of 4.99 was found in abundance on the non-viable and non-sorted sperm membranes but was not present on the membranes of viable spermatozoa. This protein was identified as an intra-acrosomal, non-bacteriolytic, C lysozymelike protein, termed SLLP1. This protein is exposed during the acrosome reaction, and retained on the equatorial segment of the sperm membrane. The absence of this protein in the viable, sorted sperm population suggests that sex-sorting actively selects sperm that are acrosome-intact. These results provide evidence that the sorting process selects a superior, homogenous population of membraneintact spermatozoa. This may explain why sorted spermatozoa survive longer in the female tract than non-sorted spermatozoa and can provide acceptable fertility rates with low-dose inseminations 1 . This work was supported by XY Inc and the Major National Research Facilities Program (Biomedical Node of the Australian Proteome Analysis Facility). 1 de Graaf, S, et al. (2007) Reprod Dom Anim 42: 648-653 P367 The effect of VEGF on the change of P44/p42 MAP kinases, Protein Kinase C and Protein tyrosine kinases of ovine oocytes matured in vitro Hailing, LUO 1 *, Xin, CAO 1 ,2, Ping, ZHOU 3 , Youzhang, ZHAO 2 , Guoqing, SHI 3 1College of Animal Science and Technology, China Agriculture University, Beijing 100094,P.R China; 2 College of Animal Science and Technology, Gansu Agricultural University, Lanzhou 730070, P.R China; 3 Xinjiang Academic of Agriculture and Reclamation Science, Shihezi, Xinjiang, 832000, P.R China P44/p42 MAP kinases (Erk1 and Erk2) and Protein Kinase C (PKC) are mediate essential cellular signals required for activation, proliferation, differentiation and survival. Protein Tyrosine Kinases (PTKs) perform a critical role in signal transduction pathways that control cell proliferation, differentiation, metabolism and apoptosis. Our previous results proved that Vascular Endothelial Growth Factor (VEGF) is a powerful mediator for vessel permeability and could improve the quality of ovine oocyte maturation in vitro. In this study, to investigate the effect of VEGF on the change of phosphorylation ERKs, PKC and PTKs activity in ovine oocyte, 5 ng/ml VEGF was supplied in media during maturation in vitro and the method of ELISA was used to determine the ERKs, PKC and PTKs activities. The results shown that the levels of phosphorylation ERKs, PKC and PTKs activity in VEGF groups were higher than the without-VEGF groups. Moreover VEGF was strongly enhanced the level of phosphorylation ERKs and PKC activity but reduced PTKs activity during the period of ovine oocyte maturation in vitro. The phosphorylation levels of ERKs and PKC were slowly reduced from 0 hour to germinal vesicle breakdown (GVBD), subsequently phosphorylation ERKs level rapidly rose when GVBD was commencing and kept this higher level to MII phase, specially arrived at top at 18 hours and 21 hours. Phosphorylation PKC level took on fluctuant change but rose little by little and maintained high level at MII phase, reached peak at 21 hours. The ERKs and PKC activity reached the highest at 21 hour in vitro culture and descended again from 21 hours to 24 hours but interestingly that the levels were mount up again in the VEGF group at 24 hours compared to the control. The level of PTKs activity rapidly lessened from 0 hour to GVBD, fluctuant changed and kept low level during the residual period of oocyte maturation, but reached high level at 21 hours. Protein tyrosine kinase activity is often associated with membrane receptor protein tyrosine kinases such as VEGFR. Phosphorylation of ERKs and PKC by PTKs is essential for the regulation of oocyte maturation biological mechanisms. It could hypothesis that activation of VEGFR-mediated pathways occurs by supplying VEGF in ovine oocyte maturation medium, which cross-link the VEGFR on the plasma membrane and initiate receptor-mediated signaling pathways, leading to ERKs and PKC activation by PTKs activity change. In conclusion, VEGF induce the activation of ERK and PKC functions dependently of the activation of PTKs, strongly supporting the view that VEGF could enhance the ability of oocyte maturation. Keywords VEGF, p44/p42 MAP kinases, Protein Kinase C, Protein Tyrosine kinases, ovine oocyte The present study was supported by National Natural Science Foundation of China (No. 30371035) P368 Mucin Gene Expression in the Equine Reproductive Tract Maischberger, E 1 *, Irwin, J 1 , Cummins, C 1 , Duggan, V 1 , Carrington, S 1 , Corfield, A 2 , Reid, C 1 1Veterinary Sciences Centre, University College Dublin- School of Agriculture, Food Science and Veterinary Medicine, Ireland; 2 Mucin research group, University of Bristol, United Kingdom Mucins, which are the principal gel-forming components of the mucus gel, play an important role in lubricating, hydrating and protecting mucosal surfaces. In particular, they contribute to a barrier against microbial infection, toxins and other potentially harmful elements in the supramucosal environment. We hypothesize that there are changes
16 t h International Congress on Animal Reproduction Poster Abstracts 149 in the composition of the mucous layer of the endometrium/cervix of mares with Persistent Post-Breeding Endometritis (PPBEM)/Ascending Placentitis (AP), which affect normal barrier function at these sites. Both of these conditions cause considerable economic losses to the Irish equine bloodstock industry. PPBEM manifests as a failure to clear semen, exogenous contaminants and bacteria from the endometrium after breeding, leading to an embryotoxic environment and failure to conceive. In AP, the cervical mucus plug is breached by invading microorganisms late in pregnancy, inducing abortion or premature birth. In an attempt to address this hypothesis, our initial objective has been to determine the expression of mucin genes within the reproductive tract of the mare. The expression of the mucin proteins is encoded by approximately 20 genes and is under the influence of steroid hormones during the oestrus cycle. We surveyed 16 mucin genes of the normal equine vagina, cervix, endometrium and uterine tube during oestrus (n=6) and dioestrus (n=12). Orthologues of human mucin genes were identified in the horse genome and their expression determined by PCR and agarose gel-electrophoresis. MUC1, a membrane-bound mucin, and MUC2, a secreted mucin, are expressed in the human and equine reproductive tract. MUC3 appears to be expressed in the equine endometrium only during dioestrus, while MUC5B is expressed only during oestrus. MUC5AC is one of the secreted mucins that are expressed in very low concentrations in the human reproductive tract and it is expressed in the equine lung. However, it is not expressed in the equine reproductive tract. MUC7, which is not expressed in the human reproductive tract, is expressed in the mare. This study provides baseline data for normal mares and for future comparison with mares affected by PPBEM or AP. Future work will include real time PCR and in- situ hybridisation to determine gene expression levels and tissue distribution within the reproductive tract throughout the oestrus cycle in healthy and diseased mares. P369 Endometrial expression of IGF-I, IGF-II and IGF-1R throughout the cow oestrous cycle Meikle, A 1 *, Carriquiry, M 2 , Chalar, C 3 , Sanguinetti, C 3 , Abreu, C 3 , Crespi, D 1 , Cavestany, D 1 1Laboratory of Nuclear Techniques, Veterinary Faculty of Uruguay, Uruguay; 2 Agronomy Faculty, Uruguay; 3 Sciences Faculty, Uruguay The insulin-like growth factor (IGF) system is expressed in bovine uterus during the estrous cycle and early pregnancy and plays an important role in regulating the development of the embryo and uterus. IGF-I and -II mediate their effects through the type 1 IGF receptor (IGF-1R). In this study, the expression of the IGFs and IGF- 1R was determined on endometrial transcervical biopsies collected on days 0 (oestrus), 5, 12 and 19 of the cow oestrous cycle (n = 8). The abundance of mRNA of IGF-I, IGF-II, IGF-1R, and an endogenous control (ribosomal protein L19; RPL19) was measured by quantitative realtime RT-PCR using SYBR Green. Abundance of mRNA of target genes was normalized to RPL19 and expressed in relative amounts to an external control (ΔΔCT -method). Results were analyzed with a repeated measures analysis (PROC MIXED of SAS) and considered to differ when P < 0.05. The expression of RPL19 mRNA did not throughout the oestrus cycle. Endometrial expression of IGF-I mRNA was the greatest at oestrus and day 5 (100%), and decreased to 47% and 35% of the initial values on days 12 and 17, respectively. Abundance of IGF-II mRNA peaked at day 12 and decreased sharply thereafter (to one third of day 12 values). Interestingly, IGF-1R mRNA expression showed the same pattern during the oestrous cycle that IGF-II mRNA. IGF-1R mRNA showed reduced expression at oestrus and day 5, increased by 1-fold on day 12, and decreased on day 17 to values similar to the oestrus ones. These results show that these IGF system components are distinctively regulated during the oestrous cycle suggesting that modulation of IGF system may influence uterine activity during this period. The earlier and later increases found on IGF-I and IGF-II respectively, suggest a differential role of these hormones on the early/late blastocyst and/or on the regulation of uterine function. The increase in the uterine sensitivity to IGFs during the late luteal phase – by IGF-1R expression- may reinforce the role of IGF-II during the early pregnancy. P370 Regulation of Lefty2 in the oviduct of cyclic, pregnant, and pseudopregnant rats. Argañaraz, ME 1 , Valdecantos, PA 2 , Miceli, DC 1 * 1Instituto Superior de Investigaciones Biológicas, CONICET, Argentina; 2Facultad de Bioquímica,Cátedra de Biología Celular, Universidad Nacional de Tucumán, Argentina Transforming growth factor beta superfamily members are closely associated with tissue remodeling events and reproductive processes, being involved in the embryo development and in the maternalembryo cross-talk. Moreover, transforming growth factor beta and their receptors were found in the preimplantation embryo and the reproductive tract (oviduct and uterus). Lefty2 an unusual member of this family has been implicated in the regulation of other transforming growth factor beta members such as nodal, activin, bone morphogenic proteins and transforming growth factor beta 1 via cryto co-receptors and by an antagonic mechanism. To date, the presence and regulation of Lefty2 in the rat oviduct have not been described yet. The aim of the present study was to investigate the expression of Lefty2 and its co-receptor (crypto) in the oviduct. RNA; oviductal proteins and tissues were collected from cyclic non-pregnant, pregnant, and hormonally-induced pseudopregnant rats. Lefty is a pre-proprotein of 42 kDa that is proteolytic activated to a mature form of 26 kDa. Lefty2 proteins were detected by western blots in the oviduct of cyclic, pregnant, and pseudopregnant rats but were not influenced by the estrous cycle. During early pregnancy, Lefty2 mature form was significantly higher at day 4 (when the embryo is still in the oviduct) and then it was gradually decreased while the pre-proprotein had not significant modifications. During pseudopregnancy, both form of Lefty2 protein were found at very low levels, without variations. Crypto transcripts, analyzed by semi-quantitative RT-PCR, were detected in the oviduct in the three studied conditions. Crypto expression levels were increased at day 4 of pregnancy, as we have reported for the lefty2 protein. Neither the estrous cycle nor the pseudopregnancy showed variation in the crypto gene expression pattern. These results suggest that Lefty2 and crypto are present in the rat oviduct during pregnancy; that Lefty2 could act along a paracrine pathway by binding to specific receptors on oviductal cells and that their expression could be independent of steroid regulation. The secretion of Lefty2 could be important for the embryo maintenance during its pass through the oviduct. P371 Oog1 is a maternal effect gene required for the development of mouse preimplantation embryo Minami, N 1 *, Imaichi, H 1 , Tsukamoto, S 2 , Ohta, Y 3 , Kito, S 3 , Kimura, K 4 , Imai, H 1Lab. Reproductive Biology, Kyoto University, Japan; 2 Physiology and Cell Biology, Tokyo Medical and Dental University, Japan; 3 Research Center for Radiation Safety, National Institute of Radiological Sciences, Japan; 4 Animal Breeding and Reproduction Research Team, National Institute of Livestock and Grassland Sci, Japan Previously we identified an oocyte-specific gene, Oog1 (Minami et al., 2001). The expression of Oog1 starts at E15.5 in embryonic ovary and continues until the 2-cell stage. The expression is dramatically degraded thereafter. The most interesting characteristic of Oog1 protein is nuclear accumulation during the late 1-cell to early 2-cell stage (Minami et al., 2003). The period coincides with the time of zygotic gene activation and the time of first mitosis. The molecular biological feature of Oog1 is the association with small GTP binding proteins, such as Ras and Ran (Tsukamoto et al., 2006). However, the precise mechanism of Oog1 in embryonic development remains unknown. In addition, since Oog1 is multicopy gene, knockout approach can not be used. In the present study, we examined the role of Oog1 in the development of mouse embryos using transgenic RNAi approach. Two constructs differing in the sequence of Oog1 inverted repeats (IR1 and IR2) were employed in this study. To make
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Reproduction in Domestic Animals - Facultad de Ciencias Veterinarias

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