bluuebunny/crossref_metadata_2025 huggingface dataset:
255 GENE EXPRESSION OF Cox 5a, 5b, AND 6b1 AND THEIR ROLES IN PRE-IMPLANTATION OF MOUSE EMBRYOS
Authors: Lee S.-E. , Li X.-Y. , Cui X.-S. , Kim N.-H.
Published: December 2006

Despite clear evidence of regulation of mitochondrial respiration by nuclear encoded genes, cytochrome oxidase (Cox), little information is available on their expression and functional roles during early embryonic development. To examine the role of Cox in oocyte maturation and embryogenesis, we first characterized mRNA and protein levels of nuclear encoded genes, Cox 5a, 5b, and 6b1, in mouse oocytes and during early embryogenesis, using real-time RT-PCR and immunocytochemistry. We then examined the possible role of these genes in oocyte maturation and pre-implantation development using RNA interference analysis. The relative abundances of Cox 5a, 5b, and 6b1 transcripts was measured by real time RT-PCR. After normalization by comparison to histone H2a mRNA levels, the mRNA expression of Cox 5a, 5b, and 6b1 were found to be considerable in mature oocytes and zygotes, but reduced slightly in 2-cell embryos. From the 2-cell to the blastocyst stage, mRNA expression is dependent on the number of blastomeres, as expression increases only gradually with development. Immunocytochemical studies revealed that Cox 5a, 5b, and 6b1 proteins were expressed in all blastomeres of the blastocyst. Injection of Cox 5a, 5b, or 6b1 siRNA into GV stage oocytes decreased expression of the target mRNA specifically, while not affecting the expression of mRNAs for the other subunits in mature oocytes. Similarly, each siRNA injection into zygotes specifically reduced target mRNA expression at the 2-cell, morula and blastocyst stages (P < 0.05). Silencing of mRNA expression by RNA interference (siRNA) did not inhibit oocyte maturation or developmental events up to the morula and blastocyst stages. The expression level of mtDNA9, as well as overall levels of mitochondrial mRNAs, was not different following injection of siRNA for Cox 5a, 5b, or 6b1. However, it is evident that the number of mitochondria in siRNA treated blastocysts was greatly reduced, and they appeared to be morphologically abnormal. Significantly higher apoptosis and lower cell numbers were observed in siRNA treated blastocysts. Real time RT PCR revealed that silencing of Cox 5a, 5b, and 6b1 decreased mRNA and protein levels of E-cadherin. These results suggest that the Cox subunits, Cox 5a, 5b, and 6b1, play an important role in mitochondrial function during pre-implantation development.
This work was funded by a grant from the National Research Laboratory Program in Korea.
257 EXPRESSION OF DEVELOPMENTAL GENES IN PORCINE NUCLEAR TRANSFER EMBRYOS RECONSTRUCTED WITH BONE MARROW MESENCHYMAL STEM CELLS
Authors: Mohana Kumar B. , Jin H.-F. , Kim J.-G. , Balasubramanian S. , Choe S.-Y. , Rho G.-J.
Published: December 2006

Abnormal gene expression is frequently observed in nuclear transfer (NT) embryos and is one of the suggested causes of the low success rates of this approach. Recent study has suggested that adult stem cells may be better donor cells for NT, as their less differentiated state may ease epigenetic reprogramming by the oocyte (Kato et al. 2004 Biol. Reprod. 70, 415-418). In the present study, we investigated the expression profile of some selected genes involved in the development of the pre-implantation embryos of in vivo- and NT-derived origin using bone marrow mesenchymal stem cells (MSCs) and porcine fetal fibroblasts (pFF) as donors. Isolated population of MSCs from porcine bone marrow were characterized by cell-surface antigen profile (CD13pos, CD105pos, CD45neg, and CD133neg) and by their extensive consistent differentiation to multiple mesenchymal lineages (adipocytic, osteocytic and chondrocytic) under controlled in vitro conditions (Pittenger et al. 1999 Science 284, 143-147). Primary cultures of pFF from a female fetus at <30 days of gestation were established. for NT, donor cells at 3-4 passages were employed. Embryos cloned from MSCs showed enhanced developmental potential compared to pFF cloned embryos, indicated by higher rates of blastocyst formation (15.3% � 4.8 and 9.0% � 3.9, respectively) and total cell number (31.5 � 7.2 and 20.5 � 5.4, respectively) in Day 7 blastocysts. Total RNA was extracted from pools (triplicates) of 10 embryos each of 8-cell, morula, and blastocyst stages of in vivo and NT origin using Dynabeads� mRNA DIRECT" kit (Dynal, Oslo, Norway). Reverse transcription was performed with a Superscript" III cDNA synthesis kit (Invitrogen, Carlsbad, CA, USA). Real-time PCR was performed on a Light cycler� using FastStart DNA Master SYBR Green I (Roche Diagnostics, Mannheim, Germany). The expression profiles of genes involved in transcription (Oct-4, Stat3), DNA methylation (Dnmt1), de novo methylation (Dnmt3a), histone deacetylation (Hdac2), anti-apoptosis (Bcl-xL), and embryonic growth (Igf2r) were determined. The mRNA of H2a was employed to normalize the levels. Significant differences (P < 0.05) in the relative abundance of Stat3, Dnmt1, Dnmt3a, Bcl2, and Igf2r were observed in pFF NT embryos compared with in vivo-produced embryos, whereas embryos derived from MSCs showed expression patterns similar to those of in vivo-produced embryos. However, Oct-4 and Hdac2 revealed similar expression profiles in NT- and in vivo-produced embryos. These results indicate that MSC-derived NT embryos had enhanced embryonic development and their gene expression pattern more closely resembled that of in vivo-produced embryos. Hence, less differentiated MSCs may have a more flexible potential in improving the efficiency of the porcine NT technique.
This work was supported by Grant No. R05-2004-000-10702-0 from KOSEF, Republic of Korea.
264 IDENTIFICATION OF THE PREDOMINANT TRANSCRIPT IN BOVINE OOCYTES, IN VITRO-DERIVED BLASTOCYSTS, AND IN NUCLEAR TRANSFER BLASTOCYSTS
Authors: Prather R. S. , Korte S. , Woods R. , Spate L. , Bivens N. , Forrester L. J. , Springer G. K. , Patel R. V. , Liu Z. H. , Murphy C. N. , Green J. A.
Published: December 2006

Identification of transcripts produced during bovine embryogenesis is the first step in describing the normal developmental program. To that end, mRNA was isolated from in vitro-matured metaphase II oocytes (MPII), in vitro-produced 2-cell-stage (2-Cell), in vitro-produced precompact morula-stage (PCM), in vitro-produced blastocyst-stage (BL), and in vitro-produced nuclear transfer blastocyst-stage (NTBL) embryos. The mRNA was isolated by using Dynabeads® (Dynal, Inc., Lake Success, NY, USA), and amplified by using the SMART system. PCR products were purified and ligated into pSPORT1 and electroporated into E. coli. Random clones were selected for DNA sequencing. Sequence data were evaluated for quality and clustered by sequence similarity with sequences generated from a larger expressed sequence tag (EST) project (http://genome.rnet.missouri.edu/Bovine/) by using the tlcluster program from the University of Iowa. Sequences over 100 bp in length with average Phred scores of over 20 for the entire sequence were submitted to GenBank (NIH genetic sequence database). Sequences were compared to the bovine TIGR (The Institute for Genomic Research) and human databases to gather annotation. The best comparison is listed below by using the HUGO Gene Nomenclature Committee standards (http://www.gene.ucl.ac.uk/nomenclature/) when possible. The number of unique clusters, i.e. no match in GenBank, was 53, 120, 109, 115, and 135, for MPII, 2-Cell, PCM, BL, and NTBL, respectively. The total number of clusters per tissue ranged from 224 to 992. The percent of clusters (number of clusters per total number of ESTs) per library was 12% (224/1762), 42% (746/1771), 48% (819/1715), 49% (900/1818) and 53% (992/1876) for MPII, 2-Cell, PCM, BL, and NTBL, respectively. Either the quality of the MPII library was lower or the complexity of the MPII mRNA was less than mRNA in the other tissues. Examples of mRNA that were in different abundance are shown in Table 1. Clearly, as in other species, there are significant changes in mRNA abundance during early embryogenesis. Furthermore, NTBL embryos, even though they are morphologically similar to BL, possess a population of mRNA that is distinct from that in BL.

Table 1.
Comparison of mRNA Abundance During Bovine Embryogenesis

This work was funded by the USDA NRI 2003–35205–12812 and Food for the 21st Century.
266 IDENTIFICATION OF Oct-4 AND Nanog, THE TWO PLURIPOTENCY MARKER GENES IN RABBIT PRE-IMPLANTATION-STAGE EMBRYOS
Authors: Tancos Z. , Kobolak J. , Baji Gal A. , Dinnyes A.
Published: December 2006

Several genes, including Oct-4 and Nanog, coordinate the embryogenesis of mammalian embryos. Whereas Oct-4 has an activator effect, the Nanog protein blocks the transcription of several genes in early stages; however, the product of these two genes appears parallel and directs early embryogenesis. The goal of this work was to isolate the Oct-4 and Nanog genes from rabbit, based on the sequences of other species published so far. The sequence of known genes has been analyzed, and primers have been designed based on similarity of sequences. Oocyte-to-blastocyt-stage embryos were collected from superovulated rabbits in RNase-free water. Embryonic mRNA was isolated by using the Dynal mRNA isolation KIT (Dynal, Biotech, Oslo, Norway). Real-time PCR was performed in a ABI PRISM� 7000 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). The reaction mixture consisted SYBR� Green PCR Master Mix (Applied Biosystems), 300 nM of each primer, and an 1/8 aliquot of the embryo cDNA in a 25-�L final volume. The cDNA template was denatured by heating to 95�C for 10 min and amplified by 45 cycles of 95�C for 15 s and 60�C for 1 min, with a single fluorescence measurement at each cycle. When the reaction was finished, melting curves were plotted to confirm product purity. The reaction mix was electrophoretically separated to facilitate fragment isolation. In the case of Nanog, a 131-bp fragment was cloned. Sequence analysis revealed significant homology with the mouse sequence. Based on this finding, new primers were designed in order to isolate a larger fragment as well as the genomic copy of the gene. In the case of Oct-4, several combinations of primers were tested, because of the rather conservative sequence of the Oct-4 transcription factor family. As a result, an optimal primer pair was found that yielded a 450 bp fragment, expected according to known sequences. After direct sequencing, there was a high similarity to Oct-4 genes, indicating that the isolated cDNA is probably part of the rabbit Oct-4 cDNA. For further analysis, the cDNA fragments of both genes were isolated (MinElute Gel Extraction Kit; Qiagen, Valencia, CA, USA) and cloned into bacterial plasmids (TOP 10 Cloning KIT; Invitrogen, Carlsbad, CA, USA). At this point, it can be stated that: (1) both genes have been successfully identified in rabbit genome, (2) a method has been developed to detect expression of genes, and (3) gene-specific primers have been produced. Our further goal is to clone the whole coding region of the genes and to identify the sequence. The cloned fragments will be used for in situ hybridization in implanted stage embryo sections.
This research was supported by EU-FP6-MEXT-CT-2003-59582, Wellcome Trust (Grant No. 070246), OTKA T046171, and NKTH BIO-00017/2002.
27 SODIUM CHLORIDE TREATMENT OF CELL MEMBRANE-PERMEABILIZED NUCLEAR DONOR CELLS FACILITATES THE DISPLACEMENT OF THE SOMATIC HISTONE H1 AND HMG-17 PROTEINS IN RECONSTRUCTED PORCINE EMBRYOS
Authors: Che L. , Bordignon V.
Published: December 2006

Developmental efficiency of somatic cell-reconstructed embryos depends on extensive remodeling of chromatin structural components. Due to their importance for maintaining the high-order chromatin structure and controlling DNA functions, including replication, transcription, repair, and recombination, histones and other chromatin-binding proteins represent leading choice markers to investigate nuclear remodeling in reconstructed embryos. The main objective of this study was to investigate whether or not the exposure of cell membrane permeabilized nuclear donor cells to sodium chloride (salt-extraction) would facilitate the displacement of chromatin-binding proteins in reconstructed porcine embryos. Both linker histone H1 (H1) and high-mobility group (HMG) proteins are known to affect gene expression through the modulation of the high-order chromatin structure. Standard methods of oocyte enucleation and electrofusion were applied for embryo reconstruction using in vitro-matured oocytes and control or salt-extracted fetal fibroblast cells. For salt-extraction, confluent cell cultures were washed with Ca2+/Mg2+-free Hank's balanced salt solution (HBSS); cells were permeabilized by incubation with 1 µg/mL of streptolysin O at 37°C for 30 min in HBSS, and then maintained in Tris-NaCl buffer (10 mM Tris-HCl, 0.5 mM MgCl2, 0.7 M NaCl, 1 M sucrose) for 5 min. After salt-extraction, cells were rinsed and cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 2 mM CaCl2 for 1 h at 37.5°C for membrane resealing prior to nuclear transfer. Reconstructed embryos were activated using ionomycin (15 µM/5 min) and strontium chloride (Sr2+; 10 mM/4 h), and then cultured in PZM-3 medium. Immunostaining for H1 and HMG-17 was performed in nuclear donor cells and embryos at different stages after reconstruction. The time required for H1 displacement in transplanted nuclei was reduced by the salt-extraction treatment (Table 1). Salt-extracted cells showed a stronger HMG-17 cytoplasmic signal compared to control cells. The proportion of HMG-17-positive reconstructed embryos at 1, 3, and 6 h was 54 vs. 19, 57 vs. 44, and 75 vs. 62, for control and salt-extracted cells, respectively. These data suggest that salt-extraction prior to nuclear transplantation enhances the remodeling of chromatin structure in embryos reconstructed with somatic cell nuclei.

Table 1.
Proportion (n) of H1-positive stained embryos after different times from parthenogenetic activation (PA) and nuclear transfer using control (NT-control) or salt-extracted (NT-extracted) cells

This work was supported by a NSERC Discovery Grant to VB.
273 APOPTOSIS EVALUATION OF IN VITRO-PRODUCED PIG EMBRYOS (PARTIAL RESULT)
Authors: Coutinho A. R. S. , Milazzotto M. P. , Peres M. A. , Marques M. G. , Nicacio A. C. , Visintin J. A. , Assumpção M. E. O. A.
Published: December 2006

Apoptosis is a physiological event involved with death and tissue replication, fulfilling an important function of tissue organizations during embryogenesis. This mechanism occurs in in vivo as well as in vitro pre-implantation embryos, but most frequently in the latter. The transcriptional activation of pig embryos occurs at the four-cell stage, which is the longer stage during the pre-implantation period. This stage is characterized by embryonic developmental blockage that decreases the production rates (embryos loss). The aim of this study was to evaluate a correlation between apoptosis mechanism and developmental blockage of IVP porcine embryos. Immature oocytes after IVM/IVF were submitted to IVC in PZM-1 medium containing BSA 3 mg/mL at 38.5�C, 5% CO2 in air and high humidified atmosphere. The embryo development was analyzed at 96 h of cultute (Day 4) in order to verify cleavage rate and blockage (4 cells) and non-blockage (e8 cells) embryo rates. Out of 625 grade I, II, and III oocytes submitted to IVP, 70.3 � 5.2% (430/625) cleaved from which 27.1 � 10.3% (166/625) were blocked and 43 � 10.8% (264/625) were non-blocked. Blocked and non-blocked embryos were assessed to evaluate apoptosis rates. Qualitative assays of embryo cells were achieved with two different DNA stains: YOPRO-1 (Molecular Probe�; Invitrogen Brasil, Ltd., Sao Paulo, Brazil), permeable though plasma membrane in the early stage of apoptosis, and TUNEL (Roche�; Amersham Biosciences, Sao Paulo, Brazil), which detects DNA fragmentation in the last stages of apoptosis. The embryos were stained with 0.1 �M YOPRO-1/mL PBS, incubated 15 min at 38�C, 5% CO2 in air and high humidified atmosphere, and immediately observed by means of confocal microscopy. For the TUNEL assay, embryos were fixed in 4% paraformaldehyde solution (w/v) in PBS for 1 h at room temperature, and incubated in permeabilization solution [0.5% (v/v) Triton X-100, 0.1% (w/v) sodium citrate in PBS] for 2 h. For positive control, samples were treated with DNase-I at 37�C for 1 h. The negative control and experimental samples were incubated with buffer solution under the same conditions. The positive control and experimental samples were incubated with enzymatic and stain solution (FITC) at 37�C for 1 h; the negative control was incubated with only enzymatic solution. The embryos were stained with Hoechst 33342 (5 �/mL) and observed by means of fluorescence microscopy. Blocked embryos showed more apoptosis (66% and 40% to YOPRO-1 and TUNEL, respectively) than non-blocked embryos (25% and 0% to YOPRO-1 and TUNEL, respectively). In conclusion, the developmentally blocked embryos suffered more apoptosis, although morphologic apoptosis assays (light and electronic microscopic) must be performed to confirm this finding.
This work was supported by FAPESP 04/01252-4.
278 EFFECTS OF LEPTIN ON IN VITRO DEVELOPMENT AND GENE EXPRESSION IN PORCINE EMBRYOS
Authors: Jeong Y.-J. , Kim J.-G. , Mohana Kumar B. , Balasubramanian S. , Choe S.-Y. , Rho G.-J.
Published: December 2006

Recent evidence suggests that leptin is an important signal in female reproduction, including control of ovarian function (Brann et al. 2002 Steroids 67, 95-104). Leptin receptor transcripts are presented in various cell types of humans, rats, and pigs. However, the results have been contradictory on the role of leptin in embryo development. The objective of this study was to determine the effects of different concentrations of leptin during porcine oocyte maturation on subsequent embryo development and expression of genes related to leptin signal transduction or apoptosis. Porcine oocytes were matured for 44 h in TCM-199 with the addition of leptin at 0 (control), 10, 50, 100, and 200 ng/mL (leptin 10, 50, 100, and 200 groups, respectively), and subsequently fertilized with frozen thawed semen (1 � 105 sperm/mL) in mTBM. Presumptive zygotes were then cultured in NCSU-23 medium. Cleavage and blastocyst rates (in seven replicates) were assessed on Days 2 and 7 (the day of IVF was defined as Day 0), respectively. The total cell number in blastocysts was counted and the number of apoptotic cells was determined by TUNEL. Expression of genes encoding leptin receptor (LEPR), cytoplasmic transcription factor (STAT3), pro-apoptotic (Bax), and anti-apoptotic (Bcl2) regulators in blastocysts were determined by quantitative reverse transcription-polymerase chain reaction (RT-PCR). The percentage of MII oocytes (in four replicates) was significantly (P < 0.05) higher in 50 and 100 groups than in the others [80 (216/270) and 84% (236/280) vs. 70 (158/224), 74 (172/230), and 75% (186/248) in the 0, 10, and 200 groups, respectively]. Oocytes matured in the 50 and 100 groups showed a significantly (P < 0.05) greater proportion of cleaved embryos than those in the other groups [82 (368/448) and 84% (380/451) vs. 71 (339/476), 76 (358/470), and 74% (327/441) in 0, 10, and 200 groups, respectively]. Furthermore, the rate of blastocyst formation at Day 7 was significantly (P < 0.05) higher in the leptin 100 group than in the other groups [29% (131/451) vs. 18 (86/476), 23 (109/470), 20 (90/448), and 22% (98/441) in control and leptin 10, 50, and 200 groups, respectively]. Increased cell number and reduced proportion of apoptotic cells were observed in blastocysts originating from the oocytes matured in the presence of 100 ng/mL leptin. Depending on the concentrations used in the maturation medium, leptin increased LEPR, STAT3, and Bcl2 mRNA levels and reduced the Bax mRNA level in blastocysts. The results indicate that addition of leptin at 100 ng/mL during oocyte maturation improved embryonic development with up-regulation of LEPR, STAT3, and Bcl2 genes and suppression of the Bax gene, and thus optimizes the in vitro culture systems for porcine embryos.
This work was supported by Grant No. 1000520040020000 from Biogreen 21, Republic of Korea.
282 IMPROVED QUALITY OF PORCINE BLASTOCYSTS BY AGGREGATION OF EMBRYOS AT THE 4 - 8-CELL STAGE
Authors: Lee S.-G. , Park C.-H. , Choi D.-H. , Son H.-Y. , Lee C.-K.
Published: December 2006

Use of blastocysts produced in vitro would be an efficient way to generate embryonic stem (ES) cells for the production of transgenic animals and the study of developmental gene regulation. In pigs, the morphology and cell number of in vitro-produced blastocysts are inferior to these parameters in their in vivo counterparts. Therefore, establishment of ES cells from blastocysts produced in vitro might be hindered by poor embryo quality. The objective of this study was to increase the cell number of blastocysts derived by aggregating 4–8-cell stage porcine embryos produced in vitro. Cumulus–oocyte complexes were collected from prepubertal gilt ovaries, and matured in vitro. Embryos at the 4–8-cell stage were produced by culturing embryos for two days after in vitro fertilization (IVF). After removal of the zona pellucida with acid Tyrode’s solution, one (1X), two (2X), and three (3X) 4–8-cell stage embryos were aggregated by co-culturing them in aggregation plates followed by culturing to the blastocyst stage. After 7 days, the developmental ability and the number of cells in aggregated embryos were determined by staining with Hoechst 33342 and propidium iodide. The percentage of blastocysts was higher in both 2X and 3X aggregated embryos compared to that of 1X and that of intact controls (Table 1). The cell number of blastocysts also increased in aggregated embryos compared to that of non-aggregated (1X) embryos and controls. This result suggests that aggregation might improve the quality of in vitro-fertilized porcine blastocysts by increasing cell numbers, thus becoming a useful resource for isolation and establishment of porcine ES cells. Further studies are required to investigate the quality of the aggregated embryos in terms of increasing the pluripotent cell population by staining for Oct-4 and to apply improved aggregation methods in nuclear-transferred (NT) porcine embryos.

Table 1.
Development, cell number, and ICM ratio of aggregated porcine embryos
284 COMPARATIVE FERTILITY OF FRESHLY-COLLECTED VERSUS FROZEN - THAWED SPERMATOZOA FOR IN VITRO FERTILIZATION IN THE FISHING CAT (PRIONAILURUS VIVERRINUS)
Authors: Magarey G. , Herrick J. , Thiangtum K. , Tunwattana W. , Swanson W.
Published: December 2006

Wild populations of fishing cats (Prionailurus viverrinus) in Southeast Asia are in decline, primarily due to habitat loss. Because the fishing cat population in North American zoos is small (n = 69) and inbred (F = 0.17) with relatively low genetic variation (86%), infusion of new founder genes from Asia is a conservation priority. Importation of cryopreserved semen for use with IVF and ET may offer one alternative to the international transport of living animals. In this study, our objectives were to (1) compare motility longevity of fresh vs. frozen-thawed fishing cat spermatozoa in two culture media, (2) evaluate ovarian responses to exogenous gonadotropins, and (3) assess development of IVF embryos produced with fresh vs. frozen-thawed spermatozoa. Raw semen was collected via electroejaculation from male fishing cats (n = 4), divided into groups, and washed. Two sperm pellets were resuspended in either Ham's F10 medium (HF10; with 5% FBS) or our feline optimized culture medium (FOCM; with 0.4% BSA); another pellet was diluted in TEST egg yolk, cooled to 5�C over 3 h, glycerated (4%), and cryopreserved in straws over LN2 vapor. Frozen sperm samples were thawed, washed, and diluted in either HF10 or FOCM. Fresh and frozen-thawed sperm motility (percent motile, rate of forward progress) in each medium (10 � 106 motile sperm/mL) was assessed (at 0, 1, 3, and 6 h) in microdrops under oil during culture (38�C; 6% CO2 in air). Female fishing cats (n = 10) were treated with exogenous gonadotropins (150 IU eCG, 100 IU hCG, 85-h interval) and ovarian follicles were aspirated laparoscopically. Recovered oocytes were inseminated with fresh (2 � 105 motile sperm/mL) or frozen-thawed (5 � 105 motile sperm/mL) spermatozoa in FOCM microdrops; resulting embryos were either cryopreserved or cultured in FOCM (with 5% FBS added at 72 h post-insemination) for 7 days. Sperm motility over time did not differ (P > 0.05) between media for either fresh or frozen-thawed samples; however, across media, frozen-thawed sperm motility was lower (P < 0.05) and declined faster (P < 0.05) compared to fresh spermatozoa. Females produced an average (�SEM) of 9.8 � 2.9 mature ovarian follicles, allowing recovery of 7.3 � 2.6 high-quality oocytes per female. Oocyte cleavage percentage at 42 h p.i. was lower (P < 0.05) with frozen-thawed spermatozoa (38%, 11/29) compared to freshly collected spermatozoa (68%, 17/25). Overall, 35% (6/17) of cultured embryos developed to blastocysts with no difference (P > 0.05) between embryos produced with frozen-thawed (4/11) vs. fresh (2/6) spermatozoa. Although fishing cat sperm motility and fertility appear compromised after cryopreservation, our results demonstrate the ability of frozen-thawed spermatozoa to produce IVF embryos that are capable of developing to blastocyst stage in vitro.
This work was supported by (NIH RR015388).
299 CONSEQUENCES OF REPEATED ULTRASOUND-GUIDED FOLLICLE ASPIRATION OF THE OVARIES OF GIR (BOS INDICUS) COWS
Authors: Viana J. H. M. , Camargo L. S. A. , Nascimento A. A. , Ferreira A. M. , Ferreira J. F. , Marques Junior A. P.
Published: December 2006

Ultrasound-guided follicle aspiration has been widely used to recover cumulus–oocyte complexes (COC) for in vitro embryo production in cattle, but the safety of this procedure is still under discussion. The aim of this study was to evaluate the negative effects of repeated follicle aspiration on ovarian structure. Pluriparous, non-lactating Gir breed (Bos indicus) cows (n = 22), previously used as COC donors, were evaluated. Follicle aspiration was performed twice a week with a portable ultrasound device, using disposable 19G or 20G needles (Neolus; Terumo Europe, Leuven, Belgium) and a vacuum pressure ranging from 60 to 80 mmHg. At the end of the collection period, cows were examined by rectal palpation and ultrasonography to estimate the degree of ovarian adherence and fibrosis. A group of cows (n = 8) punctured for different periods was selected for histological evaluation of ovarian lesions. Ovaries were surgically recovered 48 h after the last aspiration session, fixed by immersion in a solution of 4% paraformoldehyde and processed for histology. The slides obtained were stained by hematoxylin–eosin and Gomori's trichromic techniques. The association of follicle aspiration and the presence of ovarian abnormalities were estimated by Pearson's correlation method. Results are presented as means ± SEM. The cows evaluated underwent from 9 to 42 puncture sessions, and yielded from 10 to 719 oocytes. Some degree of adherence and/or fibrosis was detected in 37.5% of the ovaries. The incidence of lesions was higher in cows undergoing more than 20 OPU sessions (77.8% vs. 28.6%; P < 0.05). There was a positive correlation between the degree of ovarian adherence and the number of puncture sessions performed (R = 0.31; P < 0.05), but not with the total of oocytes recovered (R = 0.03; P > 0.05). The opposite was observed for ovarian fibrosis, which presented a positive correlation with oocyte yield (R = 0.52; P < 0.05), but not with the number of puncture sessions (R = 0.17; P > 0.05). This difference may be related to the wide range in oocyte yield per donor (means ranging from 1.1 ± 0.9 to 38.6 ± 12.8) and, consequently, in the number of punctures performed in the ovaries. Histological evaluation showed thickening of the tunica albuginea, presence of inflammatory cells infiltration in punctured areas, and increase in the proportion of conjunctive tissue in the ovarian stroma. Punctured follicles showed hemorrhagic clots and variable degrees of disorganization in the follicular wall layers, including dispersion of luteinized cells. These results show that ultrasound-guided follicle aspiration may have negative effects on ovarian structure, depending on the number of sessions performed and oocytes recovered.
This work was sponsored by FAPEMIG and CNPq.
3 NUCLEAR REPROGRAMMING OF PORCINE FIBROBLAST CELLS BY XENOPUS EGG EXTRACTS
Authors: Miyamoto K. , Nagao Y. , Minami N. , Yamada M. , Ohsumi K. , Imai H.
Published: December 2006

Much evidence indicates that somatic cells can be reprogrammed in an oocyte cytoplasm. The nuclear reprogramming consists of many unknown processes, and mechanisms underlying these processes still remain to be elucidated. Recently some reports noted that Xenopus oocytes or eggs can induce some of the reprogramming events in mammalian cells. We investigated the processes of nuclear reprogramming of porcine fibroblast cells by Xenopus egg extracts to understand how egg extracts trigger the reprogramming and/or dedifferentiation of cells. Unfertilized Xenopus eggs were collected from mature females. After removal of the jelly coat, activation was routinely achieved by calcium ionophore A23187. The eggs were immediately centrifuged and the cytoplasmic fraction was used as egg extracts. Porcine fibroblast cells were permeabilized by streptolysin O and incubated in the egg extracts under the ATP-generating system (1 mM ATP, 5 mM phosphocreatine, and 20 U/mL creatine kinase) for 30 min at 37�C or 2 h at 23�C. The incorporation of Xenopus-specific linker histone B4 into porcine fibroblasts was examined by immunofluorescence and immunobloting analysis. After collection of cells from the extracts, permeabilized membranes of the cells were resealed in culture medium containing 2 mM CaCl2 for 2 h. The cells were then incubated in DMEM with 10% fetal bovine serum (FBS) or porcine zygote medium-3 (PZM-3: Yoshioka et al. 2002 Biol. Reprod. 66, 112-119) containing 5.55 mM glucose and 5% FBS. RNAs were extracted from the cells in each culture dish and Oct-4 expression was examined by RT-PCR analysis every day until Day 8. The primers were designed to span the 99 base-pair intron region of porcine Oct-4 gene for recognizing both spliced and unspliced transcripts. The incorporation of histone B4 from Xenopus egg extracts was observed at the nuclear region of the porcine fibroblasts under both the 37�C and the 23�C conditions. Because the histone B4 incorporation was inhibited by addition of Apyrase, an ATPase, a part of reprogramming might be an ATP-dependent process. When treated cells were incubated in DMEM or PZM-3, Oct-4 expression was detected in the cells cultured in DMEM, but not in PZM-3. However, the transcripts of Oct-4 were mainly obtained in unspliced form at the earlier stage of culture (after Day 1 to Day 4 of culture), suggesting that a part of reprogramming processes by the egg extracts involves induction of dedifferention of cells or activation of a pluripotent marker gene such as Oct-4. Xenopus egg extract may provide a system to investigate the processes involving nuclear reprogramming and the pluripotent state of mammalian cells in vitro.
308 TESTICULAR DEVELOPMENT IN PREPUBERTAL JERSEY BULL CALVES IMMUNIZED AGAINST INHIBIN
Authors: Schuenemann G. M. , Mendis-Handagama S. M. L. C. , Kania S. A. , Rohrbach N. R. , Hopkins F. M. , Moorehead H. , Lunn P. , Dowlen H. H. , Schrick F. N.
Published: December 2006

Previous studies have shown that immunization against inhibin (INH) in bull calves increased subsequent sperm production (Martin et al. 1991 Biol. Reprod. 45, 73; Bame et al. 1996 Biol. Reprod. 54, 328). The objective of the current study was to evaluate the effectiveness of gonadotropin administration at initiation of inhibin immunization in bull calves on testicular morphology. The study was performed using the inhibin peptide (bovine inhibin α1–26) conjugated to keyhole limpet hemocyanin (KLH). Primary treatments administered to Jersey bull calves (initial immunization at 27 ± 5 days of age; Day 1 of the experimental period) consisted of control (KLH, 250 µg, n = 9) or immunization (INH; 500 μg INH: 250 µg KLH, n = 9) with each emulsified in 2 mL of Freund's complete adjuvant (FCA). Booster immunizations (identical preparation in FCA) occurred every 21 days with the last administration on Day 84 of the trial. Subsets of calves were randomly assigned within primary treatments (TRT) to receive saline (1 mL, n = 3/TRT), FSH (20 mg, n = 3/TRT), or GnRH (50 μg, n = 3/TRT) every 8 h (0600, 1400, and 2200 h) from Day 1 to Day 3 of the study. Blood samples were obtained daily from Days 0–14 and weekly until testes collection (Day 91) for FSH, LH, testosterone (T), and determination of antibody titers. Body weight and scrotal circumference (SC) were measured at each immunization and immediately before testes removal. The right testis was weighed and used for absolute volume calculation of cell components per testis. Tissue sections were examined using a light microscope (400×). For each cell type, absolute volume of Sertoli, Leydig, and germ cells were counted according to the point counting method. Data were analyzed using MIXED procedure of SAS (SaS Institute, Inc., Cary, NC, USA). Antibody titers were increased in INH bulls compared to KLH bulls (P < 0.05) during the experimental period. Body weight (89.8 ± 14.2 kg), SC (14.6 ± 1.3 cm), and single testicular weight (19.2 ± 6.2 g) recorded at the end of the experimental period did not differ between treatments. Neither serum concentrations of FSH, LH, and T nor population of Leydig and Sertoli cells differed between treatment groups. However, a significant immunization X hormone treatment interaction was noted for germ cell volume per testis (P < 0.008). Administration of FSH at the time of initial immunization against inhibin significantly increased germ cell population (1.22 ± 0.1 cm3) compared to INH-saline bulls (0.64 ± 0.1 cm3) with INH-GnRH bulls intermediate (0.84 ± 0.1 cm3; P < 0.05). In contrast, germ cell volume was not increased following hormone administration in KLH bulls. These results suggest that gonadotropin administration at the time of inhibin immunization increases germ cell volume in the testis without altering Sertoli and Leydig cell volume.
309 THE SPERMATOGENIC CYCLE IN MAMMALIAN TESTIS XENOGRAFTS
Authors: Zeng W. , Avelar G. F. , Rathi R. , Franca L. R. , Dobrinski I.
Published: December 2006

Grafting of immature testis tissue from different mammalian donor species into mouse hosts results in production of spermatozoa from the donor species. Xenografting of testis tissue from rhesus monkeys, pigs, and sheep accelerates sperm production. To determine whether this shortened time to sperm production is due to the reduced spermatogenic cycle length, we applied bromodeoxyuridine (BrdU) incorporation to analyze the spermatogenic cycle in porcine and ovine testis xenografts. Testes from 1-2-week-old Yorkshire cross pigs and 1-week-old Suffolk sheep were cut into small fragments (approximately 1 � 1 � 2 mm) and eight fragments were grafted under the back skin of each castrated male immunodeficient NCR nude recipient mouse (n = 7 for pig, n = 5 for sheep). Mice were given BrdU (100 mg/kg i.p.) at 7 months (porcine tissue) or 6 months (ovine tissue) post-transplantation. Mice carrying porcine tissue were sacrificed 1 h, 9 days or 18 days after BrdU injection. Mice with ovine testicular tissue were sacrificed 1 h, 11 days or 22 days after BrdU injection. Analysis time points were chosen based on the reported length of the spermatogenic cycle in pigs and sheep (approximately 9 days and 11 days, respectively). All eight stages of the spermatogenic cycle were analyzed to identify the most advanced germ cells labeled in each time period after BrdU injection. All seminiferous tubules containing full spermatogenesis were analyzed. Histologically, 51.8% (range 7 to 98%, n = 2040 tubules) of seminiferous tubules from porcine grafts, and 64.4% (range 2 to 92%, n = 2903 tubules) of seminiferous tubules from ovine grafts presented complete spermatogenesis. In porcine grafts, the most advanced germ cells labeled 1 h after BrdU injection were primary spermatocytes in pre-leptotene/leptotene at stage I of the spermatogenic cycle. At 9 days and 18 days after injection, the most advanced labeled germ cells were primary spermatocytes at pachytene at stage I and elongating spermatids at late stage II, respectively. In ovine grafts, the most advanced labeled germ cells at 1 h, 11 days and 22 days were pre-leptotene/leptotene at stage II, primary spermatocytes at the pachytene at stage I and elongating spermatids at stage II, respectively. These results indicate that each spermatogenic cycle in porcine and ovine testis xenografts lasts around 9 days and 11 days, respectively. Therefore, the length of the spermatogenic cycle is conserved in porcine and ovine testis xenografts and shortened time to sperm production is likely due to accelerated maturation of the testicular somatic components, such as Sertoli cells.
This work was supported by NIH R01 RR17359-01.
317 EFFECT OF REACTIVE OXYGEN SPECIES (ROS) ON ACTIVATION OF BOVINE OOCYTES MATURED IN VITRO
Authors: Park J. I. , Jang Y. , Lee E. S.
Published: December 2006

Vitamin E is an antioxidant that protects embryos from oxidative stress and supports embryonic development in vitro. This study was carried out to assess the effect of reactive oxygen species (ROS) by measuring the effects of hydrogen peroxide (H2O2) and vitamin E (VitE) on chemical activation of in vitro-matured oocytes. Bovine ovaries were collected from slaughtered cows at a local abattoir. Oocytes were aspirated from follicles 3-8 mm in diameter and transferred to maturation medium: tissue culture medium (TCM)-199 supplemented with 10% (v/v) fetal calf serum, 100 mg/L-cysteinr, 44 mg/L sodium pyruvate, gonadotropins (each 250 IU of eCG and hCG/mL), and 10 ng/mL epidermal growth factor. Oocytes were cultured at 38.9�C in 5% CO2 in humidified air. After 22 h of culture, oocytes with polar bodies were selected and submitted to activation treatments. Oocytes were exposed to calcium ionomycin (5 �M for 5 min) followed by incubation with 6-DMAP (2 mM) for 3.5 h in medium supplemented with or without VitE (100 �m). Routine in vitro fertilization (IVF) was also performed for 18 h as control treatment with or without VitE. After activation, oocytes were cultured in mSOF medium containing 0.8% BSA at 38.9�C in 5% CO2, 5% O2 in humidified air for 16-18 h. H2O2 content was measured and rate of development was monitored. For detection of H2O2, individual oocytes were stained with 22,72-dichlorofluorescein diacetate (DCFDA) and observed under fluorescence microscope (Hashimoto et al. 2000 Mol. Reprod. Dev. 56, 520-526). Levels of H2O2 were analyzed by counting the number of black-white pixels of TIFF images (ImageJ ver. 1.32; http://rsb.info.nih.gov/ij) after conversion of fluorescence image. Data were analyzed using Student's t-test and chi-square test. The H2O2 contents were significantly higher (P < 0.05) in the activation group without VitE (218 pixels, n = 49) than IVF without VitE (210 pixels, n = 50). Levels of H2O2 in oocytes activated with VitE were also higher (P < 0.05) than in the IVF group with VitE (216 pixels, n = 50 vs. 210 pixels, n = 51). Between groups with or without VitE, the H2O2 contents were not different in both activation and IVF. The cleavage rate to the 2-cell stage and development rate to blastocysts after activation with VitE (77.8% and 15.1%, n = 72) were significantly higher (P < 0.05) than in those without VitE (63.3% and 10.2%, n = 60). The results of the present study demonstrate that the chemical activation increased accumulation of H2O2 in oocytes compared to the IVF condition. Although ROS level did not affect chemical activation, vitamin E added in the activation medium promoted further development of activated embryos.
This work was supported by a grant from the BioGreen 21 Program (20050301-034-443-026-03-00), Rural Development Administration, Korea.
323 INTERACTIONS OF OXYGEN TENSION, GLUCOSE CONCENTRATION, AND HEAT SHOCK ON MATURATION OF BOVINE OOCYTES
Authors: de Castro e Paula L. A. , Hansen P. J.
Published: December 2006

In many cell types, oxygen tension and glucose concentration affect the magnitude of heat shock effects. Experiments were performed to (1) develop oocyte maturation protocols under high (20.95%) and low (5%) oxygen tensions, and (2) test the hypothesis that low oxygen tension and high glucose concentration reduce the magnitude of the deleterious effects of heat shock on in vitro maturation of bovine oocytes. In Experiment 1, oocytes were matured in tissue culture medium-199 (TCM-199) or synthetic oviduct fluid (SOF) containing 5.6 or 20 mM glucose, under either high or low oxygen. Fertilization was performed under high oxygen and embryos were cultured in KSOM-BE2 under low oxygen. Data were analyzed by least squares analysis of variance using the GLM procedure of SAS (SAS Institute, Inc., Cary, NC, USA), with maturation medium, glucose concentration, and oxygen tension as main effects. Oxygen concentration affected percent blastocyst formation when TCM-199 was used (greater for high oxygen than low oxygen) but not when SOF was used (medium � oxygen, P < 0.05). Similarly, glucose had no effect on percent blastocyst formation under high oxygen but high glucose was superior under low oxygen (glucose � oxygen, P < 0.05). Therefore, SOF with either 5.6 or 20 mM glucose was used to study the effect of heat shock on maturation under high and low oxygen in Experiment 2. Maturation was at 38.5�C for 22 h (control) or 41�C for 12 h and 38.5�C for 10 h (heat shock). Fertilization was performed at 38.5�C under high oxygen and embryos were cultured in KSOM-BE2 under low oxygen at 38.5�C. Data were analyzed as described above with glucose concentration, temperature and oxygen tension as main effects. The experiment was replicated nine times with a total of 3215 embryos. Heat shock decreased the cleavage rate slightly regardless of the maturation conditions (69 � 1% vs. 66 � 1%, P < 0.01). For oocytes matured in 5.6 mM glucose, percent blastocyst formation was greater for high oxygen while there was no effect of oxygen tension for oocytes matured in 20 mM glucose (glucose � oxygen, P < 0.01). Heat shock decreased the percent blastocyst formation under all maturation conditions except for the group matured under high oxygen and high glucose (temperature � oxygen � medium, P = 0.07; temperature � oxygen for oocytes under 5% oxygen; P < 0.05). The percent blastocyst formation for control and heat-shocked oocytes was 25.9% vs. 22.5% (low oxygen-5.6 mM glucose), 41.6% vs. 34.9% (low-20 mM), 41.7% vs. 35.0% (high-5.6 mM), and 37.6% vs. 37.5% (high-20 mM) (SEM = 2.0%). In conclusion, heat shock during maturation under an oxygen tension (5% oxygen) that approaches physiological conditions reduces the percent blastocyst formation. Moreover, a high glucose concentration protects oocytes from heat shock under high oxygen.
This work was supported by USDA NRICGP 2002-35203-12664 and CAPES #134202-9.
326 EVALUATION OF CULTURE DURATION AND PARTIAL CUMULUS CELL REMOVAL DURING CANINE OOCYTE MATURATION
Authors: Hanna C. , Long C. , Westhusin M. , Kraemer D.
Published: December 2006

The objectives of this study were to determine whether the percentage of canine oocytes that resume meiosis during in vitro maturation could be increased by either increasing culture duration or by removing approximately one-half of the cumulus cells 24 h after oocytes were placed into culture. Canine female reproductive tracts were collected from a local clinic and ovaries were minced in warm TL-HEPES. Oocytes with a consistently dark ooplasm and at least two layers of cumulus cells were selected, cultured in a basic canine oocyte in vitro maturation medium consisting of TCM-199 with Earl's salts, 2.92 mM Ca-lactate, 20 mM pyruvic acid, 4.43 mM HEPES, 10% fetal calf serum, 1% Penicillin/Streptomycin (GibcoBRL, Grand Island, NY, USA), and 5 μg/mL porcine somatotropin, and incubated at 38.5°C in 5% CO2 in humidified air. Treatment groups were randomly assigned and oocytes were cultured for 60, 84, or 132 h (Basic). From each of these groups, one-half of the oocytes were pipetted through a fine bore pipette to partially remove the cumulus cells 24 h after the start of culture (Basic–1/2). At the end of culture, all oocytes were denuded and the nuclear status was observed with Hoechst 33342 under ultraviolet fluorescence. All data were analyzed by ANOVA with P < 0.05. Since the canine oocyte is ovulated at the germinal vesicle (GV) stage of meiosis and requires up to five days to mature in the oviduct, it was hypothesized that an increased culture time would allow for more oocytes to undergo nuclear maturation to metaphase II (MII). It was also hypothesized that partial removal of cumulus cells would decrease the cumulus cell component in the ooplasm that sustains meiotic arrest, allowing for more oocytes to resume meiosis (RM = germinal vesicle breakdown to MII). Results within each treatment group indicate that there is no significant difference between culture duration and the percent of oocytes that mature to MII. Additionally, there was no significance in the percent of oocytes that resumed meiosis after partial cumulus cell removal. Taken together, these data suggest that neither treatment is effective in canine in vitro maturation systems, given the current maturation culture conditions.

Table 1.
Nuclear status* of oocytes for three time periods with or without
partial cumulus cell removal
334 EFFECT OF INSULIN ON IN VITRO MATURATION OF CANINE OOCYTES
Authors: Lee H. S. , Seo Y. I. , Yin X. J. , Cho S. G. , Bae I. H. , Oh D. H. , Kong I. K.
Published: December 2006

In spite of our increased knowledge of in vitro oocyte maturation techniques, the success rate of obtaining mature canine oocytes in vitro remains very low compared with that for other domestic animals. The inefficient rate of meiotic resumption of canine oocytes is probably due to both the unique reproductive cycle and inappropriate in vitro maturation (IVM) medium. In an unpublished experiment, we found that the concentration of insulin was higher in estrus bitch serum (EBS; 8833 pg/mL) than in dog follicular fluid (DFF; preovulatory follicle, 122 pg/mL), which implies its possible role in the acquisition of oocyte competence. Therefore, in the present study we investigated the effects of supplementing the IVM medium with insulin on the incidence of maturation to metaphase II. Ovaries were collected from various stages of the estrous cycle by ovariohysterectomy, and oocytes with two or more intact cumulus layers and with a diameter >110 �m were selected and used for IVM. Oocytes were cultured in modified synthetic oviduct fluid (2004 Reprod. Nutr. Dev. 44, 105-109) supplemented with 10% EBS, 20 �g/mL estradiol, and different concentrations of insulin (0, 10, 100, or 1000 ng/mL) at 38.5�C, 5% CO2 in air. After 72 h, cumulus cells were removed from around oocytes using a small glass pipette. Denuded oocytes were fixed in 3.7% paraformaldehyde supplemented with 10 �g/mL Hoechst 33342 at room temperature for 40 min. Nuclear status was observed under UV light using a fluorescence microscope. The percentage of oocytes at the metaphase II stage was not different among the four groups 6.8, 1.8, 5.4, and 2.1% in the control, 10, 100, and 1000 ng/mL insulin groups, respectively. The incidence of oocytes with pronuclear-like structures or cleaving beyond the two-cell stage was not significant higher in the 10 and 100 ng/mL insulin treatment groups than in the control and 1000 ng/mL insulin groups 20.0 and 19.6% vs. 6.8 and 6.4%, respectively. These results indicate that the addition of insulin to the in vitro maturation medium of dog oocytes had no effect on the incidence of meiotic maturation to metaphase II, nor did it affect the frequency of occurrence of spontaneous oocyte activation.