Reproduction in Domestic Animals - Facultad de Ciencias Veterinarias

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16 t h International Congress on Animal Reproduction 150 Poster Abstracts transgenic constructs containing IR1 or IR2, each inverted repeat was transferred to pRNAi-ZP3 cassette (Stein et al., 2003), to produce pZP3-oog1IR1 or pZP3-oog1IR2. In these transgenic constructs, the expression of Oog1 dsRNA hairpins is controlled by ZP3 promoter which directs oocyte-specific expression. To analyze the phenotype of the transgenic females, each founder females (C57BL/6J) was mated with the same four wild type males (C57BL/6J) in a random sequence. Successful mating was verified by detecting vaginal plug. Mated females were then housed separately for observation and recording the number of pups delivered per litter, which was averaged to assess fertility. To test the suppressive effect of dsRNAs, fertilized embryos were collected from transgenic F1 females mated with wildtype males 24 h after eCG injection. Fertilized embryos were cultured in KSOM medium and their development was recorded at 24 h interval until 96 h after eCG. At the time of embryo collection, GV stage oocytes were collected from the same female ovaries to examine the amount of Oog1 mRNA by real-time RT-PCR. We obtained nine transgenic founders and four female transgenic lines out of 5 were sub-fertile. Embryos recovered from some transgenic F1 females mated with wild-type males exhibit developmental block in culture. These results suggest that Oog1 functions in early embryo development in mouse. This approach provides a powerful method to study the function of the genes transcribed during oogenesis and early embryogenesis. P372 Cloning and sequence analysis of Sheep fertilin β and its tissue expression Narenhua, N 1 *, Fu, L 1 , Li, N 1 , Bao, XRG 2 1College of Animal Science and Medicine, Inner Mongolia Agriculture University, China; 2 Life Science of College, China Fertilin β is member of the ADAMs (metalloproteinase-like, disintegrin-like, cysteine-rich) protein family and is expressed on the sperm surface where it has been proposed to play a role in mammalian fertilization. Inhibition of the sperm-egg binding and sperm-egg fusion make fertilin an attractive target for development of an immunocontraceptive vaccine. This study examined fertilin β gene activity in relation to fertilization in the ram testis. Mixed primers for the polymerase chain reaction (PCR) were designed based on the high sequence homology of selected regions of known bovine fertilin β gene. PCR-amplified cDNA fragments generated by 3\' and 5\' rapid amplification of cDNA ends (RACE) were combined to generate fulllength cDNA sequence here for the first time. The 2217 bp cDNA has an open reading frame encoding 738 amino acids, with a molecular mass of ~82 KDa. The deduced amino acid sequence showed identity at equivalent regions of bovine (87.1 %), porcine (73.2 %), mouse (37.6%), human (58.1%), and macaque (58.0%). Bovine fertilin β contains a domain with homology to disintegrins, snake venom proteins that bind to integrins via an integrin-binding domain containing the tripeptide RGD. This partial inhibition of fusion with RGD peptides prompted the cloning of the sheep homologue of bovine fertilin β to determine if it possessed the tripeptide RGD or different amino acid sequence in its disintegrin domain. The disintegrin domain of sheep has the tripeptide TDE (instead of RGD) in its cell recognition region. In the present investigation we also report RT-PCR and in situ hybridization studies that show that the sheep fertilin β is transcripted only in adult ram testis and not in the all 3 epididymal regions. In situ transcript hybridization shows the transcript to be localized in round and enlongating spermatids in the seminiferous epithelium. The work confirms that fertilin β is expressed tissue specifically. Key word: fertilin β/disintegrin/RGD peptide/binding and fusion P373 Changes in serine/threonine phosphorylation associated to the achievement of “in vitro” capacitation in boar spermatozoa Ramió-Lluch, L* and Rodriguez-Gil, JE Unit of animal Reproduction, Dept. Animal Medicine and Surgery, School of Veterinary Medicine, Autonomous University of Barcelona, Bellaterra, Spain Several studies in different species have shown that tyrosine phosphorylation of sperm proteins is a suitable marker of the capacitation. Furthermore, capacitation has been linked to changes in the spatial location of tyrosine-phosphorylation. However, there is little information about changes on both serine- and threoninephosphorylation status during sperm capacitation. Thus, the main aim of this study has was the observation of putative changes in both expression and location of serine- and threonine phosphorylation during "in vitro" capacitation (IVC) of boar sperm. For this purpose, boar sperm from fresh ejaculates were incubated in specific capacitation medium durin 4 hours at 39ºC in a 5%CO2 atmosphere . Sperm aliquots were taken at 0, 1, 2 , 3 and 4 hours after incubation and both the general presence and spatial location of serine- and threonine-phosphorylation were evaluated. Our results showed that boar sperm had an specific pattern of phosphorylation in both serine and threonine residues. Weight of the main bands were around 25-30 KDa, 30-35 KDa and 50 KDa in serine-phosphorylation, and around 30 KDa, 50 KDa and 75 KDa in threonine-phosphorylation. IVC induced an observable rise in the expression of serine phosphorilation during capacitation, specially in the band around 30-35 KDa in both cases. These results were accompanied with specific changes in the spatial location of both serine- and threonine-phosphorylation in spermatozoa. All these results indicate that IVC is associated with specific changes in protein phosphorylation, in tyrosine residues and in both serine- and threonine residues as well. Finally, the observed spatial changes in protein phosphorylation indicates that they could be involved in the achievement of a feasible IVC. P374 Sperm DNA fragmentation and presence of varying levels of protamine 1 mRNA in Ram and Goat Roy, R 1 *, Gosalbez, A 1 , Lopez-Fernandez, C 1 , Arroyo, F 1 , García-Hurtado, J 1 , Casado, S 2 , De La Torre, J 1 , Gosalvez, J 1 1Biology, Universidad Autonoma de Madrid, Spain; 2 Research And Development, Halotech Sl, Spain Sperm DNA fragmentation has been the subject of numerous studies because the incidence of a high rate of nuclei containing damaged DNA is highly correlated with a loss of fertility. However, the origin of DNA fragmentation is mostly unknown, although apoptosis, oxidative stress, or persistence of DNA breaks produced during chromatin protamination in spermiogenesis, could be direct causes of chromatin damage. The aim of the present investigation was to analyze the correlation between different levels of protamine 1 mRNA in sperm cells from ejaculated semen samples in ram and goat with the dynamics of DNA fragmentation after ejaculation and sperm extension. For this purpose, 10 rams from Castellana breed and 10 goats all showing Sperm DNA Fragmentation (SDF) index below 6% were assessed for SDF by using the Sperm Chromatin Dispersion test (SCD; Halomax). The dynamics of SDF was assessed at T0,T1,T4 and T24 (numbers indicates hours after ejaculation in sperm samples extended and incubated at 37ºC). SDF Values were plotted against increasing incubation times. Results showed that differences in the dynamic range of SDF do exist among different animals; each animal reached a SDF index 50% of DNA fragmentation at different times (ranging from 4 to 24 h). Simultaneously, the amount of protamine 1 mRNA from mature spermatozoa was analyzed using real-time PCR. Interestingly, when levels of protamine 1 mRNA was compared with the dynamic range of SDF present in the same samples, those individuals showing a rapid increase in sperm DNA fragmentation, exhibited the lower levels of protamine 1 mRNA. These results suggest that sperm chromatin failing to achieve a proper
16 t h International Congress on Animal Reproduction Poster Abstracts 151 protamination in condensing chromatin, facilitates DNA breakage. It is suggested that a reduction in the level of deoxyribonucleic acid protection, render the DNA molecule more sensitive to external damaging agents. P375 GnRH-a induced steroid hormone receptor regulation in bovine endometrium Singh, R*; Pretheeben, T; Perera, R; Rajamahendran, R Animal Science, University of British Columbia, Canada Introduction Ovarian steroids consistently influence the endometrium and maintain its cyclicity by acting through their corresponding receptors. Estrogen receptors(ER α and ER β) and progesterone receptors (PR) are present in bovine endometrium in follicular and luteal phases of the estrous cycle, bovine ovaries and placentomes. We, most recently demonstrated the presence of GnRH receptors (GnRH-R) in bovine endometrium at both mRNA and protein level and localized these receptors to endometrial epithelial cells in both the phases of the estrous cycle. Additionally GnRH-R mRNA is also present in normal and carcinogenic human endometrium and endometriosis, where GnRH acts in an apoptotic and antiproliferative manner. GnRH is widely used in the bovine reproductive management including estrous and ovulation synchronization, induction of ovulation, post partum cyclicity, treatment of cystic ovarian disease, to overcome early embryonic mortality, and increase pregnancy rates; but there is clear lack of information on its local modulatory role in the endometrium. We find the co-existence of GnRH-R and steroid hormone receptors as interesting and there are prior reports about ligand independent activation of steroid hormone receptors. Whether GnRH through its receptors could regulate these receptors in normal endometrium is still not known and this study, for the first time examined the GnRH induced regulation of ER α and ER β and PR in bovine endometrium. Materials and Methods Uteri belonging to follicular and luteal phases of the estrous cycle were collected from the local abattoir, transported to lab within one hour. One hundred mg of endometrial explants were cultured at 37 0 C, 5% CO 2 in humidified atmosphere. After 20 h incubation, explants were treated with different doses of GnRH agonist – buserelin (0, 200, 500, 1000 ng/mL respectively), GnRH antagonist – antide (500 ng/mL) and a combination of antide (500ng/mL) and buserelin (200ng/mL) for 6 h. Two µg of total RNA extracted from each treatment was reverse transcribed using commercially available kit and the mRNA levels of ER α, ER β and PR were assessed by semi-quantitative RT-PCR and using the gene specific primers G3PDH was used as an internal control in the experiments. Optical intensity of individual bands was analyzed by Scion imaging beta and statistically analyzed by comparing to control and using student t test. Results This study revealed that GnRH (200ng/mL) upregulated ER α mRNA in both follicular and luteal phases of the estrous cycle and it this effect was more pronounced (P≤ 0.05) in the luteal phase; whereas mRNA levels of ER β and PR were not altered. Conclusions GnRH induced upregulation of ER α could have potential implications on reproductive process such as gamete transport, fertilization, cellular proliferation, uterine receptivity, implantation and maintenance of normal physiological status of the uterus and increases our understandings of the molecular basis of the reproduction at the endometrial level. P376 Effect of pre-incubation of male and female gametes with fibronectin prior to fertilization in vitro in cattle Thys, M 1 *; Nauwynck, H 2 ; Maes, D 1 ; Van Soom, A 1 1Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Belgium; 2 Department of Virology, Parasitology and Immunology, Faculty of Veterinary Medicine, Ghent University, Belgium Carbohydrates and glycoproteins modulate various adhesion and binding events in reproductive processes, including sperm-oviduct adhesion, sperm-egg interaction and embryo implantation. When fibronectin (Fn) – an extracellular matrix glycoprotein – is supplemented to the fertilization medium, a substantial inhibition of sperm penetration during bovine in vitro fertilization (IVF) was observed. To identify whether Fn interacts with either male or female gametes, 2 experiments were conducted incubating either sperm cells or cumulus oocyte complexes (COCs) with Fn prior to IVF. To evaluate the effect of Fn on sperm, 2 groups of in vitro matured bovine COCs were fertilized in standard medium. One group was inseminated with spermatozoa (1x10 6 sp/ml) previously incubated with 500 nM Fn for 30 min. The second group was fertilized with spermatozoa (same ejaculate) incubated with standard medium. Two extra experiments – where the sperm cells were incubated for 2 h resp 4 h – were performed to evaluate effect of time of sperm preincubation on inhibition of sperm penetration. To assess the effect of Fn on the female gamete, in vitro matured COCs were divided into 2 groups. The first group was fertilized under standard conditions, the second one was treated with 500 nM Fn for 30 min prior to IVF. Subsequently, a similar setup was applied on zona-free oocytes. Twenty hours after insemination, all presumed zygotes were fixed, stained with Hoechst 33342 and evaluated by fluorescence microscopy for sperm penetration and fertilization. Differences in fertilization and penetration percentage were analyzed by binary logistic regression (SPSS 15.0). Pre-incubation of sperm cells with Fn significantly decreased the sperm penetration compared to that of the control (75.2% vs 87.0%) resulting in an inhibition of penetration of 13.6%. The same tendency was observed for fertilization with or without Fn pre-incubated sperm (68.6 % vs 78.2 %). Prolonging the duration of sperm pre-incubation caused more prominent inhibition of penetration (22.2% after 2 h; 42.8% after 4 h). When pre-incubating COCs with Fn, penetration was not significantly reduced (76.2% vs 83.0 %) compared to that of the control, nor was the fertilization rate (67.3% vs 75.4%). Furthermore, Fn pre-incubation of zona-free oocytes did not affect sperm penetration (42.0% vs 46.9%) nor fertilization (37.1% vs 37.0%). In conclusion, Fn inhibits sperm penetration in bovine COCs through interaction with the sperm cell. To elucidate the underlying mechanism, identification of receptors for Fn on bovine sperm is required. P377 Effect of replacer of fetal calf serum in the development and gene expression in bovine embryos in vitro cultured Serapião, RV 1 *; Boité, MC 1,2 ; Camargo, LSA 1 ; Polisseni, J 1 ; Viana, JHM 1 ; Folhadella, I 1 ; Sá, WF 1 ; Fonseca, FA 4 1Laboratório de Reprodução Animal, Embrapa Gado de Leite, Brazil; 2Faculdade de Medicina Veterinária, Universidade Federal Fluminense, Brazil; 3 Laboratório de Genética Molecular, Embrapa Gado de Leite, Brazil; 4Laboratório de Reprodução Animal, Universidade Estadual do Norte Fluminense, Brazil Introduction The period of post fertilization embryo culture is the most critical affecting blastocyst quality. Knockout SR (Gibco Labs., Grand Island, NY) is a serum replacer optimized to support embryonic stem cells in culture and can also be used to replace serum during culture of bovine embryos. The expression of genes associated to stress response, such as heat shock proteins (Hsp), can be affected by in vitro culture conditions, including culture medium components. The aim of this study was to evaluate the effect of Knockout TM SR on
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Reproduction in Domestic Animals - Facultad de Ciencias Veterinarias

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