bluuebunny/crossref_metadata_2025 huggingface dataset:
131 DYNAMICS OF HISTONE H3 METHYLATION AT POSITIONS K4 AND K9 IN MOUSE, RABBIT, AND BOVINE PRE-IMPLANTATION EMBRYOS
Authors: Lepikhov K. , Yang F. , Wrenzycki C. , Zakhartchenko V. , Niemann H. , Wolf E. , Walter J.
Published: December 2006

In mammals, upon the penetration of sperm into the oocyte, the paternal genome undergoes dramatic epigenetic changes. Protamin packaging of DNA is replaced by histones that acquire specific modifications. In mouse zygotes, paternal DNA gets rapidly demethylated by an active mechanism. In bovine zygotes the methylation from paternal DNA is erased only partially, and in rabbit zygotes it persists at the initial level. To understand whether these reprogramming differences are also reflected in histone modifications, we examined the dynamic changes of histone H3 methylation at positions K4 and K9 in mouse, bovine, and rabbit zygotes and in preimplantation embryos using an immunofluorescence staining procedure (Lepikhov and Walter 2004 BMC Dev. Biol. 4, 12). In zygotes, maternal chromatin contains both types of histone H3 methylation. After fertilization protamines in sperm are very quickly replaced by histones. After the formation of nucleosomes, histone H3 acquires methylation at position K4 in a stepwise manner: first as mono-methylated form and later as tri-methylated. In the late zygote, both paternal and maternal pronuclei show equal levels of histone H3 methylation at position K4. Regardless of the differences in DNA reprogramming in these 3 species, H3/K9 di-methylation is not detected on paternal genomes and is only associated with maternal genomes. During the subsequent cleavage stages, H3/K9 di-methylation decreases gradually and becomes hardly detectable in 4-cell bovine and rabbit embryos. In mouse embryos, it is detectable through all the stages. Bovine embryos reacquire this type of modification at the 8-16 cell stage, and it remains at the very low levels in rabbit, embryos until the blastocyst stage. In conclusion, mouse, rabbit and bovine zygotes show similar patterns of H3/K4triMe and H3/K9diMe distribution despite the difference in paternal DNA demethylation. The dynamics of H3/K9diMe distribution patterns in cleavage stage embryos from all embryos do not correlate with embryonic genomic activation events.
132 DIFFERENCES IN RESPIRATION RATES BETWEEN IN VIVO- AND IN VITRO-PRODUCED BOVINE EMBRYOS
Authors: Lopes A. S. , Madsen S. E. , Ramsing N. B. , Larsen L. H. , Greve T. , Callesen H.
Published: December 2006

In vitro-produced (IVP) bovine embryos differ (e.g. morphology and physiology) from their in vivo counterparts. Oxygen consumption is an indicator of the overall metabolic activity of a single embryo. Therefore, the aim of this study was to determine and compare respiration rates of in vivo- and in vitro-produced bovine day 7 embryos. Diameters of these two embryo types were also compared. In vivo embryos (n = 28) were recovered from 8 superovulated Holstein Frisian cows on day 7 following AI, while IVP embryos (n = 160; Holm et al. 1999 Theriogenology 52, 683-700) were used on day 7 after fertilization. Embryos were measured (outer diameter) and morphologically evaluated (Quality 1 to 4, IETS Manual, 1998). Only transferable in vivo embryos were used (i.e. excluding Quality 4). Respiration rates were measured on each embryo by Nanorespirometer technology (Lopes et al. 2005 Reprod. Fertil. Develop. 17, 151). Data were analyzed using Proc Mixed, and values are presented as mean � SEM. Values with different superscripts differ significantly (P < 0.05). The average respiration rates were 0.82 � 0.06a nL/h for in vivo vs. 1.37 � 0.06b nL/h for IVP embryos. The average respiration rates for the different morphological qualities were as follows (nL/h, numbers in brackets): IVP: 2.1 � 0.08a (38), 1.37 � 0.07b (55), 1.08 � 0.07c (48) and 0.62 � 0.11d (19) for Quality 1, 2, 3, and 4, respectively. In vivo: 1.17 � 0.21b,c,e (6), 0.80 � 0.15c,d,e (12), and 0.64 � 0.16d,f (10) for Quality 1, 2, and 3, respectively. The average diameter (mm) of in vivo and IVP embryos was 0.157 � 0.002a and 0.176 � 0.002b, respectively. Respiration rates were directly related to embryo diameter; larger embryos were associated with higher respiration rates (y = 17.55 � 1.32 nL/h � mm, n = 188). Respiration rates of in vivo embryos were significantly lower than those of IVP embryos, regardless of quality. This difference could reflect an effect of the culture conditions on IVP embryos because media components affect embryo metabolism. Moreover, the different ages (day 7 for IVP vs. approximately Day 6.5 for in vivo embryos, because in vivo embryos are less than 7 days after fertilization at recovery) and stages (IVP: up to expanded blastocyst stage; in vivo: morula or early blastocyst stage) could have influenced the results and also partly explain the smaller diameter of the in vivo embryos. Finally, respiration rates decreased proportionately to the morphological quality within embryo type, indicating that morphological differences are reflected at the physiological level. In conclusion, this study further outlines metabolic differences between in vivo and IVP bovine embryos. Whether such differences are a manifestation of metabolic stress associated to the separation from the natural environment or reflect suboptimal culture conditions is yet to be determined.
ASL is supported by FCT, Portugal.
133 ROLE OF CASPASE-9 AND STAGE OF DEVELOPMENT IN INDUCTION OF APOPTOSIS BY HEAT SHOCK AND TUMOR NECROSIS FACTOR-α IN BOVINE PRE-IMPLANTATION EMBRYOS
Authors: Loureiro B. , Brad A. M. , Hansen P. J.
Published: December 2006

Heat shock and tumor necrosis factor-α (TNF-α) can increase apoptosis in bovine embryos in a developmental-dependent manner. It was hypothesized that addition of the caspase-9 inhibitor, z-LEHD-fmk, would block induction of apoptosis caused by heat shock of 41°C and TNF-α. Furthermore, it was hypothesized that the magnitude of induced apoptosis would increase with stage of development. Embryos were collected on day 4, 5, and 6 after in vitro insemination and were cultured for 24 h in the presence of either 100 μm z-LEHD-fmk reconstituted in 0.5% (v/v) dimethyl sulfoxide or vehicle dimethyl sulfoxide at either (1) 38.5°C for 24 h (control), (2) 41°C for 15 h followed by 38.5°C for 9 h, or (3) 38.5°C for 24 h with 10 ng/mL murine TNF-α. Embryos were then fixed, and the proportion of blastomeres undergoing apoptosis was determined using TUNEL labeling. Heat shock did not increase the percentage of blastomeres that were TUNEL-positive (% apoptosis) at day 4 (n = 100 embryos total). In contrast, heat shock increased % apoptosis at day 5 and day 6 (P < 0.04) and this effect was blocked by z-LEHD-fmk (temperature × inhibitor, P < 0.04). At day 5, % apoptosis in the absence and presence of z-LEHD-fmk was 3.8 ± 1.9% and 3.7 ± 1.7% at 38.5°C vs. 8.9 ± 1.5% and 4.1 ± 1.7% at 41°C (n = 75 embryos total). At day 6, % apoptosis in the absence and presence of z-LEHD-fmk was 3.6 ± 1.1% and 3.7 ± 1.2% at 38.5°C vs. 11.1 ± 1.1% and 6.1 ± 1.2% at 41°C (n = 168 embryos total). Mean cell number at the end of culture ranged from 21 to 26 cells at day 4, 43 to 73 cells at day 5, and 101 to 114 cells at day 6. Treatment with TNF-α also increased apoptosis at all days (P < 0.01), and z-LEHD-fmk blocked this effect (TNF × inhibitor, P = 0.05; n = 361 embryos total). Across days, % apoptosis was 3.6 ± 1.4% (control), 3.3 ± 1.3% (inhibitor), 11.1 ± 1.3% (TNF-α), and 6.0 ± 1.4% (TNF-α + inhibitor). Mean cell number at the end of culture ranged from 21 to 27 cells at day 4, 59 to 74 cells at day 5, and 105 to 115 cells at day 6. In conclusion, activation of caspase-9 dependent pathways is involved in the induction of apoptosis by heat shock and TNF-α. Moreover, the magnitude of induced apoptosis increases as embryos advance in development.
This work was supported by USDA Grant No. 2004–34135–14715 and BARD Grant No. US–3551–04.
134 BRAHMA-RELATED-PROTEIN 1 OVEREXPRESSION IN PARTHENOGENETIC PORCINE EMBRYOS
Authors: Magnani L. , Cabot R.
Published: December 2006

The chromatin remodeling protein Brahma-related-protein 1 (Brg1) has been implicated in several cellular processes that affect chromatin structure and transcription. Brg1 is a member of the SWI/SNF family, a large group of chromatin remodeling proteins, all of which have the characteristic of utilizing the energy from ATP hydrolysis to catalyze the repositioning of nucleosomes on DNA templates. Various members of this class of proteins are known to be differentially expressed throughout vertebrate development. We hypothesize that the balance of chromatin remodeling factors present in cleavage stage porcine embryos has an impact on embryonic developmental potential. As a first step to testing this hypothesis, we attempted to overexpress a wildtype version of Brg1 in cleavage stage porcine parthenogenic embryos. The Brg1 construct was a gift from Dr. Anthony Imbalzano (University of Massachusetts, Worster, MA). The Brg1 cassette ligated into the NheI site of pIRES2-eGFP, Clontech. This construct was sequenced to verify the orientation. Oocytes were matured in vitro in defined culture medium (TCM199 supplemented with 0.1% polyvinyl chloride, 10 ng/mL epidermal growth factor, 0.5 IU/mL LH, 0.5 IU/mL FSH, 1 mm cysteine) for 42-44 h at 39�C in 5% CO2. Following in vitro maturation, oocytes were denuded and activated by electroporation and cultured in NCSU23 supplemented with BSA (4 �g/mL) at 39�C in 5% CO2, 5% O2. For microinjection, activated oocytes were divided into 3 treatment groups 3-5 h after activation. Oocytes in Treatment 1 were microinjected with pIRES2-eGFP-Brg1 vector; oocytes in Treatment 2 were microinjected with pIRES2-eGFP vector; and oocytes in Treatment 3 were not injected with any construct. Approximately 40 pL were injected at a concentration of 1 mg/mL. Following injection, lysed embryos were discarded, and the intact embryos were placed in embryo culture medium for 6 days. At this time, embryos were stained with Hoechst 33342, and the number of nuclei for each embryo was determined. Expression of the injected construct was determined by the presence of GFP 24 h following microinjection. There was a tendency for embryos that developed from oocytes injected with the Brg1 construct to have increased cell numbers at day 6 of embryo culture, as compared with the other 2 groups (21.4 � 15, n = 12; 17.7 � 9.6, n = 7; 16.8 � 5.82, n = 24 for treatments 1, 2, and 3, respectively; P = 0.3). Data were analyzed using GLM procedures in SAS; embryos with cell number e8 were analyzed. There was no difference in fragmented embryos among the 3 groups. The results come from 5 independent replicates. To the best of our knowledge, this is the first time that a chromatin remodeling protein has been overexpressed in cleavage stage porcine embryos. Further experiments using an ATPase defective subunit will be performed to further examine the role that ATP dependent chromatin remodeling processes play during porcine embryogenesis.
135 THE EFFECT OF DIFFERENT TREATMENTS OF PORCINE EJACULATED AND EPIDIDYMAL SPERMATOZOA ON ROS GENERATION
Authors: Matás C. , Sansegundo M. , Ruiz S. , Gadea J.
Published: December 2006

The production of pig embryos in vitro is still relatively inefficient compared with results obtained with oocytes matured and fertilized in vivo. The main reasons for this limited performance are polyspermy after IVF and the poor developmental ability of embryos produced by IVM-IVF (Kikuchi et al. 2002 Biol. Reprod. 1033-1041). Between factors affecting polyspermy are the sperm procedures before IVF. Usually, these procedures including centrifugations that increase reactive oxygen species (ROS) formation in spermatozoa. ROS play an important role in sperm physiology including capacitation. Physiological concentrations of ROS have been proposed to enhance sperm capacitation by increasing cAMP synthesis and by inhibiting protein tyrosine phosphatases whilst activating tyrosine kinases. In general, epididymal spermatozoa appear to be able to capacitate and fertilize eggs in vitro much more easily than ejaculated spermatozoa (Yanagimachi Mammalian Fertilization. In: The Physiology of Reproduction, Raven Press 1988; 135-182). In this study, we investigated how different sources (ejaculated spermatozoa vs. epididymal spermatozoa) and sperm capacitating methods, usually employed in porcine IVF, could be affect ROS generation. Sperm-rich fractions from five fertile boars and sperm from five different epididyimides were used. The semen samples were then: (i) washed in Dulbecco's phosphate-buffered saline (DPBS) supplemented with 0.1% BSA, (ii) left unwashed, or (iii) washed on a Percoll (Pharmacia, Uppsala, Sweden) gradient (Mat�s et al. 2003 Reproduction 125, 133-141). Production of ROS was measured by incubating the spermatozoa in the in vitro fertilization medium (TALP) in the presence of 0.7 �m 22,72-dichlorodihydrofluorescein diacetate at 37�C under 5% CO2 in 100% humidified air. Every 15 min (from 15 to 135) the samples were analyzed and evaluated by flow cytometry. Measurements were expressed as the mean green intensity fluorescence units and it was used as index of ROS generation (Gadea et al. 2005 J. Androl. 26, 396-404). ANOVA analysis revealed a significant effect of sperm treatment on the ROS generation (P < 0.001). The highest value was obtained in sperm washed on a Percoll gradient and the lowest in unwashed semen. When ejaculated vs. epididymal semen was analyzed, the same tendency was observed in both. However, the values were always lower in epididymal semen than in ejaculated semen (P < 0.001). As a conclusion, ROS generation is different between treatments and between semen procedures for the time interval studied, and this finding may help to explain the different outcome in IVF among laboratories.
This work was supported by Ministerio de Educaci�n y Ciencia, AGL2003-03144.
136 FUNCTIONAL ANALYSIS OF RHOPHILIN-2 GENE IN PRE-IMPLANTATION MOUSE EMBRYO
Authors: Matsuoka T. , Sono Y. , Matsumoto K. , Amano T. , Mizuno S. , Hosoi Y. , Saeki K. , Iritani A.
Published: December 2006

Zygotic gene activation (ZGA), which starts at the G2 phase at the 1-cell stage (Latham 1999), promotes the reprogramming of gene expression and is critical for the subsequent development of pre-implantation embryos. We have investigated the molecule function of many gene clusters, DD clones obtained by Differential-Display assays for ovulated eggs at the M II stage, and 1-cell embryos at the G2 phase. The differential expression of rhophilin-2 shown in DD assays was also confirmed by 3 independent real-time PCR analyses (P < 0.05). For these reasons, in this study, we focused on the rhophilin-2 gene, which regulates cytoskeletal organization (Peck et al. 2002). At first, we identified a protein that interacts with the Rhophilin-2 protein by a yeast 2-hybrid system. To confirm the interaction between Rhophilin-2 and the putative protein obtained by a yeast two-hybrid system, we used a co-immunoprecipitaion assay. We also investigated the expression profiles of rhophilin-2 and the transcripts of the identified protein in ovary and pre-implantation embryos using real-time PCR and immunofluorescence (IF) analysis. The ICR mice at 48 h after PMSG priming were primed with hCG, and ovaries were collected at 7 h after hCG priming. Pre-implantation embryos were collected at 1-cell, 2-cell, and 4-cell stages, and cDNA was produced by mRNA isolated from 10 oocytes or embryos in each group and was subjected to real-time PCR using a TaqMan Probe system (ABI). Sectioned ovaries and pre-implantation embryos were analyzed by IF analysis using antibody of Rhophilin-2 and the identified protein. This is the first report that GABA receptor-association protein (GABARAP) was identified as a protein that interacts with Rhophilin-2, as a result of using the yeast 2-hybrid system and subsequent co-immunoprecipitation assay. After fertilization, transcript levels of rhophilin-2 significantly decreased from the 1-cell stage to the 2-cell stage (P < 0.05), but transcript levels of GABARAP significantly increased from the 1-cell stage to the 2-cell stage (P < 0.05). The IF analysis revealed localization of Rhophilin-2 and GABARAP at the nucleolus of all follicle stage in the ovary. Moreover, Rhophiln-2 and GABARAP were found to be localized on the microtubules of 1-cell and 2-cell embryos, but no signal of Rhophilin-2 was detected in 4-cell embryos. These results suggest that Rhophilin-2 protein regulates the cytoskeletal organization in 1-cell to 2-cell embryos and is involved in the molecular mechanism of cell division by coupling with GABARAP.
This study was supported by a Grant-in-Aid for the 21st Century COE Program of the Japan Mext and by a grant for the Wakayama Prefecture Collaboration of Regional Entities for the Advancement of Technology Excellence of the JST.
14 SYNCHRONIZATION OF OVULATION IN ANESTROUS NELORE COWS TREATED WITH HORMONAL PROTOCOL WITHOUT PROGESTERONE OR PROGESTAGENS
Authors: Souza A. F. , Pinheiro V. G. , Ereno R. L. , Barros C. M.
Published: December 2006

Postpartum anestrous cows are usually treated with progesterone/progestagen protocols in order to induce ovulation and sustain pregnancy. In the present work, the efficiency of a protocol (modified GPE), in which temporary calf removal (TCR) and/or administration of eCG, replaced the use of progesterone/progestagens, was evaluated in anestrous animals. Anestrous Nelore cows (40 to 60 days postpartum, n = 22) were randomly allocated to two groups: GPE/eCG and TCR/GPE/eCG. At a random stage of the estrous cycle (D0), animals from Group GPE/eCG were treated with GnRH (50 �g, licereline, i.m., Gestran Plus�; Tecnopec, Sao Paulo, Brazil) and seven days later (D7) they received PGF2� (150 mcg, d-cloprostenol, i.m., Prolise�) and eCG (300 UI, i.m., Novormon�). On D8, estradiol benzoate (EB, 1 mg, Estrogin�; Tecnopec) was administered, and 30 to 36 h afterwards all cows were inseminated at fixed-time (FTAI), without estrus detection. The animals from group TCR/GPE/eCG received the same treatment described above, but with temporary calf removal (during 48 h) before beginning hormonal treatments. The ovaries were examined by ultrasonography (Aloka SSD 500, 7.5 MHz probe) before (D-12, D-2), during (D0, D7, D8), and after (D9, D10, D11, D17, D60) hormonal treatments. The absence of CL, during the ultrasonography performed before starting the treatments, was the criterion used to classify the cows as in postpartum anestrus. Administration of GnRH (D0) induced ovulation (determined by the presence of CL on D7) in 6 of 11 cows (54.5%) from Group GPE/eCG and TCR/GPE/eCG. In relation to synchronization of ovulation after EB administration, in Group GPE/eCG, two cows (18.1%) ovulated until 12 h after FTAI, and four (36.3%) 12 h afterwards (24 h after FTAI). In Group TCR/GPE/eCG, one cow (9%) ovulated approximately 6 h before FTAI, two (18.1%) 12 h after FTAI, and two (18.1%) 12 h afterwards. Preovulatory follicles had a diameter of 11.0 � 1.3 and 11.5 � 2.6 mm in Groups GPE/eCG and TCR/GPE/eCG, respectively. Pregnancy rates, determined by ultrasonograpy 40 to 46 days after FTAI, were 27.2% (3/11) and 45.4% (5/11), respectively. The results indicate that association of TCR with protocol GPE/eCG did not improve synchronization of ovulation or pregnancy rates (P > 0.005). However, increase in pregnancy rates was observed in another experiment with a large number of animals (Pinheiro et al. 2005 Reprod. Fert. Dev. 17, 161, abstr), in which cows from Group TCR/GPE/eCG had higher pregnancy rates (51.5%; 34/66) when compared to animals from Group GPE/eCG (28.3%, 21/74; P < 0.05).
This work was supported by FAPESP and by fellowships for A. F. Souza and V. G. Pinheiro from CNPq, Brazil.
140 IMMUNOLOCALIZATION OF INDOLEAMINE 2,3-DIOXYGENASE IN PLACENTA FROM BOVINE CLONED CONCEPTUS PREGNANCY
Authors: Oliveira L. J. , Monteiro J. M. , Oloris S. C. , Rodrigues M. R. , Okada S. S. , Verechia F. V. T. , Meirelles F. V. , Visintin J. A. , Campa A. , Kfoury Jr J. R.
Published: December 2006

The catabolism of tryptophan by indoleamine 2,3-dioxygenase (IDO) activity is strongly correlated to maternal-fetal tolerance in hemochorial placentation. Several studies have been conducted on the role of IDO on maternal-fetal tolerance in human and mice placental tissue. Changes in IDO expression and activity are related to fertility problems in humans and lower IDO activity was found in women who had recurrent abortions. In mice, IDO was localized in blastocyst, cytotrophoblast, placental macrophages, stromal cells and invasive trophoblast cells, but not in the syncytiotrophoblast. Also, IDO staining was detected on villous trophoblast cells; probably due to the fact that this is the site where fetal antigen is most exposed to the maternal immune system during pregnancy. Our previous studies in bovine showed that IDO activity is present throughout pregnancy, increasing as it develops. IDO staining can be observed at the maternal-fetal interface, in the trophoblast, especially in giant cells where it increases as gestation progresses. IDO staining in the uterine epithelium remained constant during gestation and absent in the fetal mesenchyma. This study aimed to qualitatively immunolocalize IDO staining in placenta from bovine cloned conceptuses at term. Fibroblasts originated from one bull were cultured and fused with enucleated oocytes to produced clone embryos. Placentomes were collected from four cloned conceptuses and 10 normal conceptuses (control) at parturition. Samples were fixed in 10% buffered formalin and embedded in paraffin. Sections were made and processed for immunohistochemistry using a commercial monoclonal antibody against IDO. Placenta from two cloned conceptuses showed IDO staining on fetal mesenchyma, differing from normal pregnancy where the staining of this enzyme was absent in all control samples. IDO staining was absent at the fetomaternal interface in one sample from cloned placenta and present in all normal pregnancy animals. It was difficult to establish a staining profile for giant and binucleate cells in cloned placenta, since IDO staining varied greatly among individuals. IDO immunolocalization in placenta from cloned conceptus was altered in all animals studied. It may raise the question that cloning could interfere in maternal immune modulation during pregnancy and IDO misexpression in fetomaternal tolerance could be involved in a decreased maintenance of cloned pregnancies.
141 EXPRESSION OF GENE PRODUCTS ENCODING CNN PROTEINS DURING THE FIRST WEEK OF DEVELOPMENT
Authors: Pin A. , Watson A.
Published: December 2006

CCNs (cysteine-rich 61 (Cyr61), connective tissue growth factor (CTGF), and nephroblastoma overexpressed (Nov)) are a newly characterized gene family encoding 30-40 kDa proteins that are cysteine-rich and associated with the extracellular matrix. CCNs bind integrins and activate intracellular signaling, resulting in kinase activation and gene transcription. They are involved in events essential to development, including implantation and placentation. For example, CTGF is increased during decidualization and both CTGF and Cyr61 are produced by trophoblast giant cells. Furthermore, Cyr61-null mice display early embryonic lethality due to placental defects. Although CTGF protein has been reported in mouse 4.5-day embryo, no studies to date have focused on defining the expression of CCN mRNAs during pre-implantation development. The objective of this study was to characterize the expression of CCN mRNAs during the first week of in vivo mouse and in vitro cow development. CD-1 mice were superovulated by i.p. injection of pregnant mare serum gonadotrophin (10 U) and human chorionic gonadotrophin (10 U) 22 hours later. Following mating of the mice with CD-1 males, embryos were flushed from oviducts and uteri at 2-cell, 4-cell, 8-cell, morula, and blastocyst stages. Cow oocytes were obtained from slaughterhouse ovaries. Cow embryos were produced by aspiration of early antral follicles followed by in vitro oocyte maturation, fertilization, and embryo culture methods, and collected at 2-5-cell, 6-8-cell, morula, and blastocyst stages. Groups of five embryos of each stage were pooled for mRNA extraction using a magnetic bead protocol. Reverse transcription-polymerase chain reaction was used to detect CCN mRNAs at each time point, and product identity was verified by sequencing. CCN mRNAs are detectable during the first week of development in both species. In the mouse, Cyr61 mRNAs were present in all embryo stages investigated. CTGF mRNAs were variable at the 2-cell stage, but were consistently present from the 4-cell stage to the blastocyst stage. Interestingly, Nov appeared transiently at the 4- to 8-cell stage only. In the cow, CTGF and Cyr61 mRNAs were also detectable. Nov was undetectable in the cow. This is the first report of CCN mRNA expression in mouse or cow pre-implantation development. The early appearance and continued presence of Cyr61 and CTGF indicate a specific role in early development. The transient nature of Nov expression may indicate a specific role during compaction in the mouse. The expression of CTGF and Cyr61 in the cow indicates that these proteins are conserved between species and supports the hypothesis that they are important in this early phase of development.
This research was funded by NSERC, Canada.
142 INTRACELLULAR CALCIUM UPTAKE AS A RESPONSE TO DIFFERENT CAPACITATION TREATMENTS IN EJACULATE AND EPIDIDYMAL BOAR SPERMATOZOA
Authors: Sansegundo M. , Ruiz S. , Gonzalez A. , Atucha N. T. , Matás C.
Published: December 2006

Both cauda epididymal and ejaculated spermatozoa are considered fully mature, although these two types of spermatozoa do not necessarily show the same behavior in vitro. It has been reported that both types of spermatozoa fertilize eggs in vitro at the same rate, but, in general, epididymal ones achieve this objective easier than the ejaculated ones. Intracellular Ca2+ influx is one of the crucial biochemical events occuring capacitation and Ca2+ requirements for capacitation varies among different species. In this study, we investigated how different source of spermatozoa (ejaculated vs. epididymal) and commonly employed sperm capacitating methods can affect calcium uptake. Sperm-rich fractions from seven fertile boars and sperm from seven different epididymides were used. Semen samples were kept unwashed (method A), washed in Dulbecco's phosphate-buffered saline (DPBS) supplemented with 0.1% BSA (method B), or washed on a Percoll gradient (method C) (Mat�s et al. 2003 Reproduction 125, 133-141). To measure intracellular free Ca2+, spermatozoa, treated as described above, were incubated with 2.5 �m fura-1/AM in a non capaciting medium (Tardif et al. 2003 Biol. Reprod. 68, 207-213) for 45 min at 37�C. Then, spermatozoa were resuspended in TALP medium, incubated (5% CO2, 38.5�C) for a further 60 min and then analyzed in a fluorescence spectrofluorometer with excitation wavelength set at 340-880 nm and emission held at 510 nm. The calculation of intracellular free Ca2+ was performed according to the equation of Grynkiewicz et al. (1985 J. Biol. Chem. 260, 3440-3450). Results showed that calcium uptake is affected by treatment and semen source (P < 0.001). The intracellular free Ca2+ concentrations (nM) in ejaculated semen and in epididymal spermatozoa were 269.52 vs. 208.52, 1134.58 vs. 137.37 and 1224.79 vs. 216.54 for A, B, and C methods, respectively. As a conclusion, it can be stated that sperm capacitation treatment affects calcium uptake. In addition, capacitation pathways may be modified or regulated in some way by seminal plasma, thus increasing intracellular calcium levels in ejaculated sperm (methods B and C) in comparison to those in epididymal spermatozoa.
This work was supported by Ministerio de Educaci�n y Ciencia, AGL2003-03144.
144 EPIGENETIC ASYMMETRY IN HISTONE H3 LYSINE 9 DIMETHYLATION STATUS IN PRONUCLEAR STAGE PORCINE EMBRYOS
Authors: Sega M. , Cabot R.
Published: December 2006

Covalent modification of specific residues on the tail regions of core histone proteins has been shown to play a key role in regulation of the genome. Methylation of lysine 9 on histone H3 (H3K9) is associated with repression of transcription and formation of heterochromatin domains. The maternal- and paternal-derived pronuclei from pronuclear stage murine embryos have an asymmetric distribution of H3K9 dimethylation between the 2 pronuclei; maternal pronuclei have the H3K9 dimethylation, whereas the paternal pronuclei lack this modification. The aim of this study was to characterize the H3K9 dimethylation pattern in cleavage stage porcine embryos. Indirect immunocytochemical staining was performed using a commercially available antibody from Upstate (Charlottesville, VA) that recognizes the dimethylated form of lysine 9 on histone H3 and fluorescein isothiocyanate (FITC)-conjugated secondary antibody on germinal vesicle (GV) stage porcine oocytes and cleavage stage porcine embryos. Germinal vesicle stage oocytes were matured in vitro for 44 h in a chemically defined maturation medium (TCM 199 supplemented with 0.1% PVA, 0.069 mg/mL cysteine, 10 ng/mL EGF 0.5 IU/mL LH, and 0.5 IU/mL FSH) at 39�C in 5% CO2. Following in vitro fertilization, presumptive zygotes were cultured in NCSU23 medium containing 4 mg/mL BSA at 39�C in 5% CO2, 5% O2. Embryos were fixed in 3.7% paraformaldehide for 2 h and washed in PBS and 0.1% Tween20. H3K9 dimethylation is present in the nuclei of GV-stage oocytes (n = 24) and pronuclear (n = 13), 2-cell (n = 4), 4-cell (n = 9), blastocyst (n = 8) stage embryos. The analysis revealed 2 interesting findings. First, examination of thin optical sections through the nuclei of processed embryos on a confocal microscope revealed that in GV-stage oocytes and pronuclear, 2-cell and 4-cell stage embryos the dimethylated form of H3K9 was distributed throughout the nuclei at these developmental time points, whereas in blastocyst stage embryos, the dimethylated H3K9 was restricted to the nuclear periphery. Second, not all pronuclei within pronuclear stage embryos were positive for the dimethylated form of H3K9. To determine if differential H3K9 methylation pattern observed among pronuclei in 1-cell stage embryos was due to parental origin, we produced parthenogenic porcine embryos by electrically activating porcine oocytes in calcium containing activation medium and processed them as indicated above for fertilized oocytes (n = 13). All pronuclei found in pronuclear stage parthenogenic porcine embryos were positive for dimethylation of H3K9. Chi square analysis revealed this pattern to be different from that observed in pronuclear embryos produced by fertilization (P < 0.05). In summary we conclude that the pronuclei in 1-celled embryos produced by in vitro fertilization are differentially dimethylated at H3K9 and that the localization of dimethylated H3K9 changes during cleavage development.
147 IDENTIFICATION OF PRIMORDIAL GERM CELLS IN PORCINE EMBRYOS FROM THE PRIMITIVE STREAK STAGE
Authors: Vejlsted M. , Offenberg H. , Maddox-Hyttel P.
Published: December 2006

In embryonic stem cell research, Oct-4 is one of the most widely used markers of pluripotency. Moreover, at least in the mouse, this marker is restricted to primordial germ cells (PGCs) after gastrulation. Vimentin is often used as a marker of mesoderm/mesenchyme in embryonic tissues and appears to localize to the same embryonic cells as Oct-4, at least in the bovine epiblast. The expression of neither of these markers has been completely addressed in the pig. Therefore, the purpose of the present study was to examine the expression of Oct-4 and vimentin in the porcine epiblast during differentiation and establishment of the three germ layers, i.e. the process of gastrulation. A total of 410 porcine embryos were collected at 8 to 17 days post-insemination from 29 sows of the Danish Landrace breed. Embryos were categorized based on stereo-microscopic observations into the following stages: pre-streak stages 1 and 2, primitive streak stage, neural groove stage, and somite stage. Specimens were fixed at all stages, dehydrated and embedded in paraffin wax. Selected embryos at each stage (n = 28) were completely cut into serial sections for immunohistochemical evaluation of Oct-4 and vimentin. Pre-streak stage 1 embryos were defined by lack of polarization of the embryonic disk, whereas in pre-streak stage 2 embryos a crescent shaped thickening was seen at the posterior pole of the disk. This thickening, marking the first morphological anterior-posterior polarization of the embryo proper, was shown to be a site of incipient ingression of cells from the epiblast. Immunohistochemical analyses localized Oct-4 to nuclei and vimentin to cytoplasm of both founding and ingressing epiblast cells. During formation of mesoderm and endoderm, at the primitive streak stage, solitary Oct-4 positive cells, i.e. potential PGCs, were seen scattered in the endoderm. Cells of the epiblast displayed positive labeling for Oct-4 until specification for the ectoderm cell lineage at the subsequent neural groove stage. In mesoderm, Oct-4 likewise disappeared by the time of formation of the first somites, defining the following somite stage. Thus, at this stage the only cells labeled for Oct-4, i.e. potential PGCs, were seen solitarily scattered in the endoderm. By the 15-somite stage, such cells were no longer visible in the endoderm but were seen located in the mesoderm, spreading from the stalk of the yolk sac and allantois and extending through the mid- and hindgut areas into the incipient genital ridge. Vimentin localized to the mesenchyme and most other derivatives of neural crest and mesodermal origin. In conclusion, based on Oct-4 labeling and distribution pattern, we strongly believe that we have identified the porcine PGCs from the primitive streak stage.
149 EXPRESSION OF ANGIOGENIC FACTORS AND THEIR MAJOR RECEPTORS IN THE SHEEP PLACENTA THROUGHOUT THE EARLY PREGNANCY
Authors: Borowicz P. P. , Redmer D. A. , Grazul-Bilska A. T. , Ptak G. , Loi P. , Reynolds L. P.
Published: December 2006

Embryonic losses are high in mammals, with more than 30% of fertilized eggs not resulting in an offspring. The development of the placenta is critical for normal fetal growth and development as it provides for exchange of respiratory gases, nutrients, and wastes between the fetal and maternal systems. Placental vascular development determines the rate of placental blood flow, which is a primary determinant of placental function. Recent studies suggest that vascular endothelial growth factor (VEGF), its receptors (VEGFR), along with angiopoietins (Ang-1 and Ang-2) and their common receptor Tie-2, are major placental angiogenic factors, along with fibroblast growth factor-2 (FGF-2) and its receptor (FGFR). To evaluate the patterns of placental expression of these factors during early placental development, gravid uteri were obtained from ewes (n = 6 per day) on Days 12, 18, 24, 30, and 40 of gestation (day of mating = Day 0). At slaughter the uterine and embryonic tissues were weighed and representative samples of utero-placenta (CAR - caruncle, maternal placenta; ICAR - intracarunclar, endometrium; FM, fetal membranes) were snap frozen on dry ice and analyzed for relative mRNA levels by real-time RT-PCR (ABI Prism 7000, Sequence Detection System, Applied Biosystems, Monza, Italy) of vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor-1 (VEGFR-1), vascular endothelial growth factor receptor-2 (VEGFR-2), angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2), receptor for both angiopoietins (Tie-2), fibroblast growth factor-2 (FGF-2), and fibroblast growth factor receptor (FGFR). The data were analyzed by nonlinear procedures using proc reg of SAS (SAS Institute, Inc., Cary, NC, USA). In CAR, the data showed the exponential increase from Days 12 to 40 in mRNA expression for VEGFR-1 (P < 0.0004; 0.04398e0.08794�day), VEGFR-2 (P < 0.01; 0.119208e0.06537�day), Ang-1 (P < 0.005; 0.00488e0.10881�day), Ang-2 (P < 0.0001; 0.01591e0.07864�day), Tie-2 (P < 0.03; 0.00488e0.06852�day), and FGFR (P < 0.08; 0.24577e0.04721�day). In the CAR, we also observed an exponential decrease in mRNA concentration for VEGF (P < 0.05; 28.193e-1.0719�day). In ICAR, we observed an exponential increase in mRNA concentration for VEGF (P < 0.05; 1.11685e0.06865�day), VEGFR-1 (P < 0.07; 0.09853e0.0383�day), Ang-1 (P < 0.09; 0.009318e0.05711�day), and Ang-2 (P < 0.004; 0.012647e0.09973�day). For FM, no changes in mRNA levels were observed from Days 12 to 40, but levels of all mRNAs were similar to those in CAR and ICAR. Based on the patterns of mRNA expression, these data indicate that these angiogenic factors may play an important role in early placental angiogenesis in sheep.
This work was supported by NIH grant HL64141 to LPR and DAR.
15 INCREASING ESTRUS DETECTION RATES WITHIN FOUR DAYS IN A PROSTAGLANDIN-BASED ESTRUS SYNCHRONIZATION PROGRAM IN BOS INDICUS BEEF (SISTANI) CATTLE
Authors: Jalinous M. D. , Niasari-Naslaji A. , Sarhaddi F. , Naghzali E.
Published: December 2006

Variation in estrous response is a major concern in estrus synchronization programs using prostaglandin F2�. The objective of this study was to confine estrous detection within two phases (48 h each) after synchronizing follicular wave emergence using ovarian follicle regressing (first phase) and ovulating (second phase) agents. Cyclic Sistani cattle (Iranian Bos indicus beef breed) at unknown stages of the estrous cycle and e58 days open were randomly assigned to two groups in mid summer. Females received a ration comprised of wheat straw (39%), Lucerne hay (28%) and concentrate (33%) that comprised 10% crude protein and energy at 2.1 Mcal/kg DM, according to NRC recommendations for beef cattle. In phase one, females in Group 1 (n = 33; heifers: 9; lactating cows: 4; dry cows: 20; 59 � 5.8 months of age; 361.2 � 11.5 kg LW; and 2.1 � 0.41 lactations) received estradiol benzoate (2 mg, i.m., Aburaihan Pharmaceutical Co., Tehran, Iran) and progesterone (100 mg, i.m., Aburaihan, Iran) followed by an injection of prostaglandin analogue (15 mg, i.m., Prosolvin; Intervet, Boxmeer, The Netherlands) and PMSG (400 IU, i.m., Folligon; Intervet) on Day 8. Females in Group 2 (n = 35; heifers: 9; lactating cows: 5; dry cows: 21; 59.3 � 5.7 months of age; 356.8 � 11.5 kg LW; and 2 � 0.35 lactations) received two consecutive injections of prostaglandin 14 days apart. In the second phase, starting 72 h after prostaglandin injection, females in both groups that did not exhibit estrus were given an intra-muscular injection of Gonadorelin (100 �g, Fertagyl, Intervet) followed by prostaglandin injection 7 days later. In both phases, standing estrus was recorded for 48 h, after prostaglandin injection, at 4-h intervals, 30 min each time. Within a defined period (48 h after prostaglandin injection) 12 h after standing estrus, females were inseminated with frozen semen. Pregnancy was diagnosed on Day 30 after insemination using ultrasonography. Data were analyzed using logistic regression and chi-square tests. There was no significant effect of parity and lactation status on the frequency of estrus and conception rates (P > 0.05). The total frequency of observed standing estrus throughout two phases for Group 1 and two females were 81.8% and 80%, respectively (P > 0.05). The conception rates from a single AI for Group 1 [1st phase: 70.6% (12/17); 2nd phase: 40% (4/10); total: 59.2% (16/27)] did not differ (P > 0.05) to that for Group 2 [1st phase: 66.7% (8/12); 2nd phase: 56.2% (9/16); total: 60.7% (17/28)]. The result of this study demonstrated that estrous detection rates of e80% can be achieved within 4 day observation period in Bos indicus beef cattle.
The authors express their great appreciations to the Intervet Company, Holland and the Aburaihan Company, Iran, for kind provision of pharmaceutical materials to perform this experiment.
150 EFFECT OF TIMELY FLUNIXIN MEGLUMINE TREATMENT ON PREGNANCY RATES IN HOLSTEIN HEIFERS
Authors: Guzeloglu A. , Erdem H. , Saribay M. K. , Thatcher W. W. , Tekeli T.
Published: December 2006

The objective was to determine if administration of flunixin meglumine (FM) to heifers following insemination would increase pregnancy rate due to its inhibitory effect on prostaglandin F2� (PGF2�) synthesis. Fifty-two 15-month-old Holstein heifers were synchronized with single or double injections of PGF2� followed by an injection of gonadotropin-releasing hormone (GnRH) 48 h later and a timed artificial insemination at 12-14 h after injection of GnRH (Day 0). Heifers randomly assigned to the treatment group (FMG) were injected twice with FM (1.1 mg/kg BW; i.m.), given 12 h apart on the evening of Day 15 and the morning of Day 16. The control group (CG) was not treated. Pregnancy rates were defined as the percentage of heifers diagnosed pregnant by ultrasound at Days 29 and 65 after AI. Effects of treatment on pregnancy rates and pregnancy losses were analyzed by chi-square test and logistic regression analysis. Pregnancy rates in the heifers treated with FM were higher at Day 29 (76.9%; 20/26 [FMG] vs. 50%; 13/26 [CG]; P < 0.04) and tended to be higher at Day 65 (69.2%; 18/26 [FMG] vs. 46.2%; 12/26 [CG]; P < 0.09). Administration of FM two times at a critical stage leading up to pregnancy recognition, associated with corpus luteum maintenance, increased both early embryo survival and pregnancy rate via an additive antiluteolytic effect due to a delay in the luteolytic secretion of PGF2�; this provided conceptuses extra time to develop the capability to inhibit the luteolytic process.
152 SURVIVAL OF BIOPSIED DAY 15 BOVINE CONCEPTI RE-TRANSFERRED TO SYNCHRONIZED RECIPIENT HEIFERS
Authors: Lee R. , Berg M. , McGowan L. , Peterson J. , Ledgard A. , Li N.
Published: December 2006

In cattle, a significant proportion of in vitro-produced (IVP) blastocysts do not result in viable pregnancies after transfer to recipient surrogates. Betteridge et al. (1980 J. Reprod. Fert. 59, 205–216) showed that it was possible after superovulation to recover elongated bovine embryos up to Day 17, transfer them into synchronized recipient cows, and have them develop further. We investigated the feasibility of recovering cattle embryos at Day 15, taking a sample of the trophoblast and transferring the embryos into recipients afterward for further development. The biopsied material could be used later to evaluate gene expression and correlate the profile retrospectively with developmental potential. With this approach, a larger amount of material is available for study and only embryos surviving to the elongation stage would be examined. In our experience, 30–40% of transferred blastocysts do not develop to the elongation stage. In three separate experiments, IVP embryos were generated using abattoir derived oocytes and cultured in SOF-aa supplemented with BSA (Thompson et al. 2000 J. Reprod. Fert. 118, 47–55). Six graded Day 7 (Day 0 = day of IVF) blastocysts were transferred into synchronized recipient heifers (n = 10 for each experiment). At Day 15 of gestation, concepti were flushed from the uteri after slaughter with EmCare Flush (ICPbio, Ltd., Suckland, New Zealand) containing 25 mg/mL kanamycin sulfate and then put into EmCare Hold. Conceptus lengths were measured and a proportion of those >30 mm long were cut off (5–15 mm) at one end and the trophoblast kept for future analysis. Pairs of cut or uncut (control) concepti were loaded into 0.25-mL embryo transfer straws. Each pair was transferred nonsurgically into recipients synchronized at Days 15 (Expt. 1, n = 17) or 13 (Expts. 2 and 3, n = 16 and 17, respectively) of the estrous cycle. The time between embryo flush and transfer to a recipient was noted. At Day 30, embryo survival was assessed at slaughter and compared using the Fisher's exact and chi-square. Day 15 conceptus lengths varied between 1 and 140 mm. The time between flush and transfer varied between 13 and 126 min and did not affect the ability of the concepti to subsequently establish pregnancies. Transfer to an earlier uterine environment did not significantly improve embryo survival. The proportion of embryos recovered at Day 30 was not affected by the biopsy. Up to 10 mm can be removed from 40–10 mm concepti without effect on subsequent survival. However, the overall survival post-Day 15 transfer is still too low for practical application.

Table 1.
155 QUALITY OF PORCINE EMBRYO PRODUCED IN TWO EMBRYO CULTURE MEDIA AS ASSESSED BY TOTAL CELL NUMBER AND TIME COURSE OF BLASTOCYST HATCHING
Authors: Agca Y. , Men H. , Mullen S. F. , Riley L. K. , Prather R. S. , Critser J. K.
Published: December 2006

The ability to produce porcine embryos of good quality will have a significant impact on a number of porcine assisted reproductive technologies, such as cloning, intracytoplasmic sperm injection, and embryo cryopreservation. However, porcine embryos resulting from current serum-free embryo culture systems differ significantly both structurally and functionally from those derived in vivo (Wang et al. 1999 Mol. Reprod. Dev. 53, 99-107). In this experiment, the quality of porcine embryos produced by North Carolina State University (NCSU)-23 medium (Petters and Wells 1993 J. Reprod. Fertil. Suppl. 1993, 48, 61-73) and porcine zygote medium (PZM)-1 (Yoshioka et al. 2002 Biol. Reprod. 66, 112-119) were compared by assessing the total cell number and the time course of in vitro blastocyst hatching. Porcine embryos were produced by in vitro maturation and fertilization using serum-free systems. After fertilization, presumptive zygotes were randomly allocated to either PZM-1 or NCSU-23 for subsequent development. On Day 4 of culture, the embryo culture media were supplemented with 10% fetal bovine serum (FBS). Day 6 blastocysts from each group were counted and the blastocysts were subsequently fixed in 4% formalin for counting the total cell number. The cell number in each embryo was determined by counting the nuclei after staining with bisbenzimide (Hoechst 33342). To assess the hatching ability of blastocysts, Day 6 blastocysts were cultured until Day 9 and hatched blastocysts were counted daily. Day 6 blastocyst rates (ratio of blastocysts to oocytes) and total cell number count were replicated three times. The time course of blastocyst hatching experiment was repeated four times. The data were analyzed using a chi-square test, Fisher's exact test, or Student's t-test. The blastocyst rate from culture in PZM-3 was 19.4 � 0.96% (mean � SEM), which was similar to that (16.7 � 3.2%) resulting from culture in NCSU-23 (P > 0.05). However, the total cell number in Day 6 blastocysts cultured in PZM-3 was significantly higher than for blastocysts cultured in NCSU-23 (57 � 3.1 vs. 46 � 1.7; P < 0.01). The total hatching rates (ratio of hatched blastocysts to total blastocysts) by Day 9 were similar between the two culture systems (50.1 � 9.1% vs. 50.7 � 4.1%; P > 0.05). However, on Day 6, 2.1% of blastocysts from PZM-3 culture hatched whereas no blastocysts from NCSU-23 culture hatched. The cumulative hatching rates from PZM-3 culture on Day 7 were significantly higher than those from NCSU-23 culture (15.1 � 3.8% vs. 2.6 � 1.1%; P < 0.01). In conclusion, these data suggest that blastocysts produced in PZM-3 medium have better quality than blastocysts produced in the NCSU-23 culture system as assessed by the total cell number and the time course of blastocyst hatching.
This project was supported by a grant from the National Institutes of Health (U42 RR 018877).