bluuebunny/crossref_metadata_2025 huggingface dataset:
29 BIRTH OF A FOAL CLONED BY ADULT SOMATIC CELL NUCLEAR TRANSFER WITH ROSCOVITINE-TREATED DONOR CELLS
Authors: Choi Y. H. , Chung Y. G. , Varner D. D. , Hinrichs K.
Published: December 2006

Only one horse foal produced from adult somatic cell nuclear transfer has been reported in the scientific literature (Galli et al. 2003 Nature 425, 680); a second foal from the same laboratory was reported in the popular press in 2005. In these reports, the blastocyst rates were 3 and 17%, and efficiency to birth of a live foal from total reconstructed oocytes was 0.1 and 0.5%, respectively. In cattle, roscovitine treatment of donor cells has been associated with a decrease in blastocyst development, but an increase in live births (Gibbons et al. 2002 Biol. Reprod. 66, 895-900). The present study was performed to determine the effect of roscovitine treatment of donor cells on blastocyst production after equine nuclear transfer and to evaluate the viability of pregnancies established via this treatment. In Experiment 1, fibroblasts were either grown to confluence or treated with 15 �g/mL roscovitine, for 24 h. Enucleated in vitro-matured oocytes were reconstructed by direct injection of fibroblasts using a piezo drill. Recombined oocytes were activated by injection of stallion sperm extract, followed by culture in the presence of 2 mM 6-DMAP for 4 h. They were then placed in culture in DMEM/F-12 with 10% fetal bovine serum (FBS) under mixed gas for 8 days and evaluated for blastocyst development. In Experiment 2, oocytes recombined with either confluent or roscovitine-treated donor cells were activated as above either alone or with the addition of 10 �g/mL cycloheximide at the time of 6-DMAP treatment. Resulting blastocysts from Experiment 2 were transferred transcervically to the uteri of recipient mares. One embryo was transferred per mare. In Experiment 1, there was no difference in rates of cleavage (73-19%) or blastocyst development between confluence and roscovitine treatments (2/55, 3.6% vs. 2/56, 3.6%, respectively). In Experiment 2, there was no significant difference in rates of cleavage (78-18%) or blastocyst development (0-1%; 4/105, 0/104, 0/106, 2/108) among donor cell or activation treatments. Six blastocysts were transferred to mares: two from confluent donor cells and four from roscovitine-treated donor cells. One mare, which received an embryo from the roscovitine donor/6-DMAP treatment, established pregnancy after transfer. The pregnancy continued normally and the mare delivered a colt with minimal assistance on Day 389. Typing for 13 equine microsatellites confirmed that the colt was of the same DNA type as the donor fibroblasts. The colt has grown and developed normally. Results of these studies show that roscovitine treatment of equine donor cells does not negatively affect the proportion of recombined oocytes that progress to the blastocyst stage. A viable colt resulted from an embryo produced with roscovitine-treated donor cells. More work is needed on methods to increase blastocyst rates after nuclear transfer in this species.
This work was supported by the Link Equine Research Endowment Fund, Texas A&M University.
292 EXPRESSION PROFILES OF STRESS AND METABOLIC MARKER GENES DURING IN VITRO PRODUCTION OF BUFFALO (BUBALUS BUBALIS) EMBRYOS
Authors: Rajhans R. , Kumar G. S. , Sharma G. T.
Published: December 2006

An increased understanding of the pre-implantation embryo developmental stage, with respect to physiological interaction of embryo with its micromilieu both in vivo and in vitro, is imperative to comprehend the events of pre-implantation development.The objective of the present study was to examine the temporal expression of heat shock protein 70 (Hsp-70) and glucose transporter 1 (Glut1) genes in pre-implantation-stage buffalo embryos produced under the standard in vitro production (IVP) system. Embryos were produced from slaughterhouse ovaries employing standard in vitro embryo production protocol, and presumptive zygotes produced following IVM/IVF were cultured in vitro in mSOF under mineral oil; FCS (10%) was added at 48 h post-insemination (hpi).The time series of development at stages being zygote (18-20 hpi), 2-1 cell (48 hpi), 8-16 cell (94-96 hpi), morula (120-144 hpi), and blastocyst (168-192 hpi), pre-implantation embryos conforming to the above developmental pattern were considered as 'fast-cleaving embryos', and all the embryos that did not conform to the above developmental timing were regarded as 'slow-cleaving embryos'. Pools of immature oocytes (IM, 120), Matured oocytes (MO, 120), 8-16 cell stages (8-16, 70), morula (M, 28), and blastocyst (B, 9) were collected and prepared for total RNA isolation and RT-PCR for the specific transcripts, with �-actin as loading control. The total RNA content ranged from 2.5 to 5.0 ng per oocyte/embryo. Presence of Hsp 70 and Glut1 gene transcripts was assessed in different stages of buffalo pre-implantation embryos using primers designed from bovine Hsp 70 and Glut1 by using the OLIGO program. RT was standardized using the embryo equivalent of 1-10 oocytes/embryo as the template as described by Arcellana-Panlilio and Schultz 1993 (Methods Enzymol. 225, 303-328) with PCR conditions being 59�C and 62�C for 45 s with 39 cycles for Glut1 and Hsp 70 gene transcripts, respectively. Amplicons were subjected to restriction digestion and sequencing (Acc. No. AJ812563, AJ812564). The expression of Hsp 70 throughout pre-implantation development in the fast-cleaving embryos indicated their maternal and zygotic origin, but transcripts of the Hsp 70 gene, represented by a single 488-bp amplicon, were not detected in slow-cleaving embryos, suggesting altered zygotic expression. Glut1 expression was prominent from the 8-16 cell stage, indicating a metabolic shift from pyruvate to glucose after the pre-compaction stage. For slow-cleaving embryos, transcripts of the Glut1 gene, represented by a single 327-bp amplicon, were absent during morula- and blastocyst-stage embryos, indicating the poor developmental competence of these embryos, which morphologically appeared normal. These transcription patterns reflect the embryonic response to the in vitro culture conditions and also correlate with the embryo quality and the speed of development of the pre-implantation buffalo (Bubalus bubalis) embryos.
293 EFFECT OF FOLLICULAR WAVE SYNCHRONIZATION ON IN VITRO EMBRYO PRODUCTION IN COWS
Authors: Ramos A. F. , Rumpf R. , Mollo M. R. , Câmara J. U. , Pivato I. , Marques Jr A. P. , Sartori R.
Published: December 2006

In order to achieve the ideal time of ovum pickup (OPU) for in vitro embryo production (IVP) in cows regarding number and quality of oocytes recovered, this study investigated the effect of synchronization of wave emergence with estradiol benzoate (EB) injected 7 days prior to follicular aspiration. In a Latin square design, 12 crossbred beef cows were randomly divided into three groups, with three replicates each. Cows were synchronized with a norgestomet ear implant for 7 days followed by an i.m. prostuglandin F2� (PGF2�) injection and aspiration of all ovarian follicles larger than 3 mm in diameter. After that, follicles from cows in group 2X were aspirated twice a week with 4- and 3-day intervals, and follicles from groups 1X and 1X-EB were aspirated once a week. Cows from group 1X-EB also received an im injection of 2 mg of EB immediately after OPU. Throughout the study cows were kept with an ear norgestomet implant that was replaced every 2 weeks. Ultrasound evaluations of numbers of follicles greater than 3 mm in diameter and size of the largest follicle at the time of OPU were performed. Recovered oocytes were evaluated for quality, and the viable ones (Grades I, II, and III) were in vitro-fertilized on Day 0. Cleavage rate was evaluated on Day 2 and blastocyst production on Day 7. Continuous variables were compared by ANOVA and binomial data were compared by chi-square. For the 2X group, only data from the OPU performed 3 days after the last OPU were used for analysis. Results are presented as percentages or mean � SEM. Size of the largest follicle was greater (P < 0.05) in 1X coes (12.9 � 0.2 mm) than in 1X-EB cows (11.1 � 0.3 mm), which was greater than in 2X (9.6 � 0.4 mm) cows. The 1X cows had more follicles at OPU than 2X cows (17.5 � 0.7 vs. 14.1 � 0.9), whereas the 1X-EB group (15.9 � 0.7) was intermediate and not different from the others. There was no difference in the mean number of recovered oocytes among 2X (9.6 � 0.6), 1X (12.7 � 0.8) and 1X-EB (12.3 � 1.0) cows, and the mean number of viable oocytes among groups (5.8 � 0.5, 7.3 � 0.5, and 7.0 � 0.6) for 2X, 1X, and 1X-EB cows, respectively). The rate of viable oocytes was also similar among groups [58.8% (191/325) for 2X, 58.4% (267/457) for 1X, and 57.2% (231/404) for 1X-EB cows]. Cleavage [68.6% (131/191), 65.2% (174/267), and 68.4% (158/231)] and blastocyst [38.7% (74/191), 43.8% (117/267), and 44.2% (102/231)] rates were also not different among 2X, 1X, and 1X-EB groups, respectively. Although the use of 2 mg of EB in association with a norgestomet implant 7 days prior to OPU altered the follicular wave profile, it was not enough to improve number and quality of the oocytes recovered. Moreover, this study failed to demonstrate a positive effect of OPU earlier after wave emergence, when the effect of dominance should be less pronounced, on IVP in cows.
The first author was supported by the fellowship 141077/2004-2 of CNPq, Brazil.
294 THE EFFECT OF PLASMIN ON THE IN VITRO FERTILIZATION ABILITY OF PORCINE GAMETES
Authors: Rhee H.-H. , Sa S.-J. , Cheong H.-T. , Yang B.-K. , Park C.-K.
Published: December 2006

Plasminogen activators (PAs) are specific proteolytic enzymes that convert the inactive proenzyme plasminogen to plasmin. The plasmin formed is a nonspecific, potent protease that cleaves blood fibrin clots and several other extracellular proteins. The purposes of the present study were (1) to assess the effect of plamin on sperm viability and acrosome reaction (AR), (2) to examine the effect of plasmin on zona pellucida (ZP) solubility and the binding of sperm to ZP, and (3) to evaluate the effect of plasmin on fertilization responses, including penetration and incidence of polyspermy during in vitro fertilization in the pig. Ejaculated semen was collected from three mature Duroc boars by artificial vagina. The same three boars were used for all experiments. The oocyte maturation medium used was North Carolina State University-23 (NCSU-23) medium supplemented with 10% (v/v) porcine follicular fluid (pFF), 0.6 mM cysteine, 10 IU/mL human chorionic gonadotropin (hCG; Sigma-Aldrich Corporation, St. Louis, MO, USA), and 10 IU/mL pregnant mare's serum gonadotropin (PMSG; Sigma). Porcine spermatozoa, which were washed in Dulbecco PBS (Sigma), were resuspended and incubated in fertilization medium (mTBM) containing 0, 0.1, 1.0, 10.0, or 100.0 ng/mL plasmin (Sigma). Data were analyzed by ANOVA and Duncan's multiple-range test using the Statistical Analysis System (SAS Institute, Inc., Cary, NC, USA). The present study suggests that sperm viability was not affected by plasmin treatment. Also, addition of plasmin in doses ranging between 0.1 and 100.0 ng/mL for 2, 4, or 6 h to washed boar spermatozoa resulted in enhancement of acrosome reaction (AR), compared with untreated cells. Concentrations of 0 and 0.1 ng/mL plasmin (83 � 15 and 95 � 18 sperm/oocyte, respectively) had no effect on sperm binding, whereas 1.0 (123 � 21 sperm/oocyte), 10.0 (124 � 16 sperm/oocyte), and 100 ng/mL (124 � 15 sperm/oocyte) plasmin increased (P < 0.05) sperm binding, compared with the control. The zona pellucida solubility (zona digestion time) was significantly (P < 0.05) lower in medium with 1.0 (123 � 24 s), 10.0 (99 � 15 s), or 100.0 ng/mL (95 � 19 s) plasmin, compared with control (176 � 27 s). When porcine oocytes and spermatozoa were co-incubated in various concentrations of plasmin for 6 h, the penetration rate was significantly (P < 0.05) higher in medium with 1.0 ng/mL plasmin (77.5 � 3.1%), compared with control. However, there were no significant differences in the polyspermic rates and mean numbers of sperm (MNS)/oocyte among the groups treated with plasmin and the control group. We found that addition of plasmin to fertilization medium increases the percentage of acrosome-reacted spermatozoa and the sperm-binding ability of the pig ZP. These results suggest that plasmin may play a role in events related to fertilization in the pig.
298 SPERM CAPACITATED WITH CALCIUM IONOPHORE AS A VECTOR FOR IN VITRO PRODUCTION OF BOVINE TRANSGENIC EMBRYOS
Authors: Simões R. , Milazzotto M. P. , Yamada C. , Feitosa W. B. , Coutinho A. R. S. , Visintin J. A. , Assumpção M. E. O. A.
Published: December 2006

Production of transgenic mouse embryos by microinjection is a well established and successful technique. However, when microinjection protocols were used for bovine, the amount of the oocyte lipid content did not allow the production of bovine transgenic embryos. Sperm-mediated gene transfer (SMGT) is an alternative for this species because it has lower cost and does not require microinjection handling. One of the procedures to introduce exogen DNA into oocytes is by means of sperm capacitated with calcium ionophore (CaI). The aim of this work was to evaluate different CaI concentrations ([CaI]), sperm incubation times with CaI (tCa), and incubation times of sperm capacitated with DNA (tDNA) (EYFP; Clontech, Palo Alta, CA, USA) to establish a satisfactory method for IVP of bovine transgenic embryos. Slaughterhouse oocytes with compact cumulus and uniform ooplasm were in vitro maturated in TCM-199 medium + 10% FCS + FSH + hCG + estradiol (E2) + piruvate + gentamicin under 5% CO2 in air, at 39�C and high humidified atmosphere for 24 h. Semen was thawed in a water bath at 37�C for 30 s and separated by Percoll gradient (45/90%) at 600g for 30 min. After this procedure, sperm cells were washed in TALP-semen medium by centrifugation at 200g for 5 min at room temperature. Supernatant was removed and capacitation (5 � 106 spermatozoa/group) was induced with CaI (250 nM or 500 nM for 1 or 5 min). Capacitated sperm cells were incubated with 500 ng/mL DNA for 1 or 2 h. Nontreated spermatozoa were used as control group. Sperm cells (1 � 105) were used to inseminate 20 oocytes/90 mL microdroplets for 18 h. The presumptive zygotes were co-cultured in SOFaa medium with a granulosa cell monolayer under high humidified atmosphere, at 39�C and 5% CO2 in air. Blastocyst rates were analyzed by ANOVA. Independent variables were replicate, [CaI], tCa, tDNA, and the double and triple interactions among the last three variables; when appropriate, means were compared by orthogonal contrasts. There was [CaI] � tCa � tDNA interaction for blastocyst rate (P < 0.02). Treatments with 250 nM ([CaI]), 5 min (tCaI), and 1 h (tDNA) or 500 nM ([CaI]), 1 min (tCaI), and 1 h (tDNA) resulted in 36.1% and 37.4% blastocyst rates, respectively, similar to the control group (30.5%; P > 0.4). These results demonstrated that it is possible to capacitate spermatozoa with CaI to produce transgenic embryos, without alteration of blastocyst rate.
This work was supported by FAPESP 03/08542-5 and 03/07456-8.
30 NUCLEAR AND MICROTUBULE DYNAMICS IN SOMATIC CELL NUCLEAR TRANSFER SHEEP EMBRYOS ACTIVATED WITH T-DIMETHYLAMINOPURINE AND CYCLOHEXIMIDE
Authors: Coppola G. , Jeon B.-G. , Alexander B. , John E. St. , Betts D. H. , King W. A.
Published: December 2006

The early reprogramming events following somatic cell nuclear transfer (SCNT) determine the fate of the cloned embryo and its development to a healthy viable offspring. In the present study, we undertook a detailed immunocytochemical study of the patterns of both microtubules and chromatin during the first cell cycle of sheep nuclear transfer embryos after fusion and artificial activation using either 6-dimethylaminopurine (6-DMAP) or cycloheximede (CHX). Sheep oocytes were collected from abattoir ovaries and matured in vitro for 18-20 h and enucleated; fetal fibroblasts were transplanted using standard SCNT techniques. Reconstructed cell-cytoplast couplets were fused and activated with ionomycin, followed by culture in two separate groups containing 6-DMAP (2 mM) or CHX (10 �g/mL) for 3 h. Following activation, embryos were cultured in in vitro culture (IVC) medium for blastocyst development. Embryos (n = 15, 3 replicates) were randomly removed from culture at various time points and stained using standard immunocytochemical methods to observe microtubule and nuclear configurations. Images were captured using laser scanning confocal microscopy. Results reveled that at 1 h post-fusion, 63.3% of reconstructed embryos underwent nuclear envelope breakdown (NEBD) and premature chromosome condensation (PCC) was apparent as chromosomes were situated on a non-polar spindle. The remaining embryos showed abnormal spindle and DNA configurations including chromosome outliers, congression failure, and non-NEBD. At 1 h post-activation (hpa), the embryos treated with 6-DMAP had already formed a clearly visible pronucleus (diameter 6-8 �m), whereas in the CHX-treated group, none of the embryos were at pronuclear stage; instead most of the latter embryos showed two masses of chromatin. At 1 hpa, 6-DMAP- and CHX-treated embryos showed one swelled pronucleus with a mean diameter of 8.4 � 1.3 �m and 25.8 � 0.8 �m, respectively (P < 0.05). At 16 hpa, embryos from both treatment groups still showed one swelled pronucleus. In the 6-DMAP-treated embryos, most of the embryos showed a metaphase spindle with aligned chromosomes of the first mitotic division as early as 18-10 hpa, whereas in the CHX-treated group embryos were still at the pronuclear stage. Typical 2-cell division was seen in most of the 6-DMAP-treated embryos between 24 and 30 hpa, but it was slightly delayed in CHX-treated embryos (32-35 hpa). Blastocyst development rates in the 6-DMAP- and CHX-treated groups were 21.4 � 5.6% and 14.0 � 6.3%, respectively (P < 0.05). In summary, artificial activating agents 6-DMAP and CHX exhibited different effects on chromatin remodeling, cell cycle progression, and the degree of pronuclear swelling which may explain the poor developmental rates and abnormal chromosome complements observed for cloned embryos.
This work was funded by NSERC, OMAF, and International Council for Canadian Studies.
303 INDUCED MOTILITY OF MESA/TESE SPERMATOZOA IS ASSOCIATED WITH INCREASED FERTILIZATION AND IMPLANTATION RATES IN HUMAN
Authors: Zheng H. L. , Fengand L. J.
Published: December 2006

The key for human IVF using the intracytoplasmic sperm injection (ICSI) procedure is to find moving and living sperm in azoospermic men. The objective of this study was to determine the effect of induced sperm motility on the fertilization and implantation rates in humans. Testicular sperm extraction (TESE) medium containing sperm stimulators and nutritional elements is specially designed for male factor and/or poor quality sperm with lower motility. Men with obstructed or absent ejaculatory ducts were treated by using a microepididymal sperm aspiration (MESA) procedure. Sperm are aspirated directly from the epididymis for use in the ICSI procedure. Men with non-obstructive azoospermia were treated by using the TESE procedure. Small samples of testicular tissue are obtained by needle or microsurgical biopsy and a few sperm are painstakingly dissected out of the tissue for use in the ICSI procedure. Sperm specimens either from epididymis or testis were divided into the two respective groups. In Group I, the specimens were washed in a TESE medium plus 10% human serum albumin (HSA), and then incubated with TESE medium containing 10% HSA in a 5% CO2 incubator to induce sperm motility. In Group II, the specimens were washed in human tubal fluid (HTF) medium plus 10% HSA, and then incubated with HTF containing 10% HSA in a 5% CO2 incubator. After 1 h of incubation, the motility of spermatozoa was evaluated under a microscope. The ICSI procedure was performed under an inverted microscope. Fertilized eggs were cultured in HTF medium containing 10% serum substitute supplement (SSS) until Day 3 for embryo transfer. Patients' pregnancy was followed after embryo transfer. This study demonstrated that incubation of spermatozoa from MESA and TESE procedures in TESE medium, as compared with HTF medium, results in significantly increased sperm progression (MESA sperm: 2-3 vs. 0-1; TESE sperm: 1-1.5 vs. 0-0.1) and motility (MESA sperm: 12.0 � 1.8% vs. 3.3 � 0.5%; TESE sperm: 4.5 � 0.2% vs. 0.15 � 0.02%) all respectively. Induced motility of spermatozoa in TESE medium, as compared with HTF medium, is associated with a significantly increased fertilization rate (MESA sperm: 74.5 � 3.5% vs. 60.2 � 3.1; TESE sperm: 73 � 4.3% vs. 48.4 � 3.7%) and implantation rate (MESA sperm: 29.8 � 3.8% vs. 19.4 � 2.7%, for patient age 32 � 3.0, in Group I, n = 13, and in Group II, n = 12; TESE sperm: 23.2 � 3.3% vs. 15.4 � 2.8%, patient age 33 � 2.8%, in Group I, n = 11, and in Group II, n = 9), and significantly decreased the ICSI performing time (30 � 10 vs. 120 � 20 min), all respectively. TESE medium can significantly increase the motility of spermatozoa from TESE/MESA, because it contains specific sperm stimulators and nutritional elements that directly resulted in the increase of fertilization and implantation rates.
305 SPERM CHROMATIN STRUCTURE AND DNA METHYLATION BULLS
Authors: Hengstberger K. , Phutikanit N. , Berking O. , D'Occhio M. , Sester D. , Carroll B.
Published: December 2006

Mammalian spermatogenesis is a complex process that involves genome-wide deprogramming and reprogramming of DNA methylation at different stages in order to produce sperm that are capable of fertilization and also TO support ongoing embryo development (Webster et al. 2005 PNAS 102, 4068-4073). Embryonic mortality occurs when there is inappropriate expression of developmentally regulated genes of both maternal and paternal origin. DNA methylation is associated with epigenetic regulation of gene expression and it was recently shown that aberrant DNA methylation causes a change in sperm chromatin structure (Webster et al. 2005). Studies, primarily in man, have indicated that sperm chromatin instability, as determined by the sperm chromatin structure assay (SCSA), does not influence fertilization but is linked with early pregnancy failure (Evenson and Jost 2000 Methods Cell Sci. 22, 169-189). The aim of the present study was to investigate the relationship between chromatin structure and DNA methylation in bull sperm. Chromatin structure was determined using the SCSA that involves the exposure of sperm to a low pH (1.2) detergent solution followed by the addition of acridine orange (AO). When exposed to a 488 nm laser, AO fluoresces green when bound to native (double-stranded) DNA and red when bound to denatured (single-stranded) DNA. The SCSA yields a value for the DNA fragmentation index (DFI), and in men a DFI > 27-30% is associated with early embryonic mortality (Larson-Cook et al. 2003 Fertil. Steril. 80, 895-902). Semen was obtained by electroejaculation from Zebu (Bos indicus) bulls (n = 12) in a subtropical environment; a proportion of bulls had a DFI < 15% (n = 4) and the remainder a DFI > 27% (n = 8). The DNA methylation pattern, as determined by the amplified methylation polymorphism (AMP) protocol (Webster et al. 2005), appeared to differ between sperm with a DFI < 15% and sperm with a DFI > 27% when assessed by principle coordinate analysis. Also, sperm with a DFI < 15% had a more consistent methylation pattern compared with apparent differences in methylation among sperm with a DFI > 27%. These preliminary findings could be interpreted to suggest that methylation status can contribute to chromatin structure in sperm of mature bulls. It remains to be determined whether the environment can influence sperm chromatin status in bulls by altering DNA methylation which then impacts on the conformational changes in DNA that occur during spermiogenesis.
This work was supported, in part, by Meat and Livestock Australia.
311 ULTRASTRUCTURAL ANALYSIS OF OVINE PREANTRAL FOLLICLES CULTURED IN THE PRESENCE OF INDOLE ACETIC ACID, EGF, AND FSH
Authors: Andrade E. , Landim-Alvarenga F. , Silva J. , Max M. , Alfieri A. , Seneda M. , Figueiredo J. , Toniolli R.
Published: December 2006

Previous studies from our team demonstrated that ovine primordial follicles are successfully activated in vitro after culturing in medium supplemented with 40 ng/mL indole acetic acid (IAA); besides that, the addition of IAA and epidermal growth factor (EGF) or EGF and follicle stimulating hormone (FSH) to the culture media were the most effective treatments to sustain the health and viability of activated ovine primordial follicles during in vitro culture. In this work, follicular quality was assessed only by histological studies; there is a great need to evaluate ultrastructural changes occurring in primordial follicles during activation in vitro. The aim of this study was to investigate the ultrastructural characteristics of preantral follicles after culturing of cortical slices in media containing IAA, EGF, and FSH, alone and in combination. Ovaries (n = 8) from adult merino ewes were collected at local slaughterhouses. Small pieces of ovarian fragments were removed for transmission electron microscopy (TEM). These pieces of ovarian cortex were cultured in culture dishes containing 1 mL aliquots of culture medium at 39�C with 5% CO2 in air. The media used were: (T1) Minimum essential medium (MEM) supplemented with ITS (insulin 6.25 �g/mL, transferrin 6.25 �g/mL and selenium 6.25 ng/mL), 0.23 mM pyruvate, 2 mM glutamine, 2 mM hypoxantine, 1.25 mg/mL bovine serum albumin (BSA), and antibiotics (100 ��g/mL penicillin and 100 ��g/mL streptomycim) (MEM+, control medium); (T2) MEM+IAA (40 ng/mL); (T3) MEM+IAA + epidermal growth factor (EGF) (100 ng/mL; Sigma, St. Louis, MO, USA); or (T4) MEM+EGF+FSH (100 ng/mL). After 6 days of culture of cortex tissue fragments in media, ultrastructural analysis was performed on preantral follicles (n = 3 in each treatment) included in a small cortical fragments. Preantral follicles were classified according to the stage of development as primordial follicles or as developing follicles. Preantral follicles cultured in supplemented media for 6 days were ultrastructurally normal. Their oocytes had an intact nucleus and cytoplasm that contained heterogeneous-sized lipid droplets, and numerous round or elongated mitochondria with intact parallel cristae were observed. Occasionally these organelles were associated with smooth endoplasmic reticulum (SER). Rough endoplasmic reticulum (RER) was rarely found. The cytoplasm of granulosa cells contained a large number of mitochondria and abundant RER. In contrast, follicles cultured in MEM+ (control) had a large number of vacuoles in the oocyte cytoplasm and excessive clustering of the chromatin material in the nucleus, suggesting an initial process of oocyte degeneration. In conclusion, the presence of IAA, EGF, FSH and their combinations helped to maintain ultrastructural integrity of ovine preantral follicles in cortical slices cultured in vitro.
315 EFFICIENCY OF CHEMICAL OR ELECTRICAL ACTIVATION OF BOVINE OOCYTES
Authors: Milazzotto M. P. , Feitosa W. B. , Simões R. , Mendes C. M. , Assumpção M. E. O. A. , Visintin J. A.
Published: December 2006

Activation of in vitro matured oocytes is essential for the success of nuclear transfer embryo production. Oocyte activation is promoted by the release of intracellular calcium and influx of extracellular ions, and can be chemically induced by calcium ionophores such as A23187 (CA) or ionomycin (IO). Electrical stimulation (EL) is an essential stage in nuclear transfer protocols for the fusion of enucleated oocytes with the donor's cell nucleus. Moreover, EL can be used as an alternative method to induce calcium influx through the formation of pores in the plasma membrane. This work aimed to evaluate the effect of electrical pulse vs the use of different calcium ionophores (A23187 or ionomycin) as primary agents of bovine oocyte activation, with or without the addition of BSA, on the rate of blastocyst formation and blastocyst quality. BSA was used to quench the activation process after a 5-min exposure to CA or IO. Cumulus-oocyte complexes were matured in TCM-199 medium with FCS and hormones for 18 h at 38.5�C and 5% CO2 in air. After removal of cumulus cells, oocytes presenting the first polar body were selected and maintained in SOFaa medium to complete 24 h of maturation. They were then divided into five treatments groups 1-CA (CA 5 mM, 5 min); 2-CAB (CA 5 mM, 5 min; BSA, 5 min); 3-IO (IO 5 mM, 5 min); 4-IOB (IO 5 mM, 5 min; BSA, 5 min); and 5-EL (EL 1.5 kV/cm, 20 �s, 2 pulses). After treatments, oocytes were kept in 6-dimethylaminopurine for 3 h and cultured in SOFaa medium for 7 days at 38.5�C and 5% CO2 in air. Rates of cleavage and blastocyst were evaluated respectively on Days 2 and 7 of culture. To evaluate embryo quality, Hoechst 33342/propidium iodide staining was used. Data were evaluated by ANOVA and submitted to LSD test for embryo rates and t-test for embryo quality. Four replicates were carried out with a total of 89 oocytes per treatment. There was a difference (P < 0.05) in rate of development to blastocyst between treatments 1-CA (54.4%a), 3-IO (51.4%a), and 5-EL (54.5%a) compared with 4-IOB (18.3%b). Treatment 2-CAB (39.8%ab) did not show any difference from the others. There was no difference (P > 0.05) among treatments in total number of cells: 1-CA (63.1a), 2-CAB (57.2a), 3-IO (60.9a), 4-IOB (72.4a), and 5-EL (58.4a). However, there was a difference (P < 0.01) in the percentage of viable cells between treatments 1-CA (49.9%a), 2-CAB (45.8%a), 3-IO (64.9%a), and 4-IOB (50.9%a) in comparison to 5-EL (82.7%b). In conclusion, BSA, when associated with IO, had a negative effect on embryonic developmental rates. The different calcium ionophores used and the BSA did not improve embryo quality. Although there were no significant differences between electrical and chemical activation on the rate of blastocyst formation, it is important to point out that higher quality embryos were achieved by using electrical activation.
This work was supported by FAPESP 03/00156-9.
319 OXYGEN CONSUMPTION OF BOVINE CUMULUS CELLS AND OOCYTES CULTURED IN DIFFERENT CULTURE SYSTEMS FOR OOCYTE MATURATION
Authors: Abe H. , Shiku H. , Aoyagi S. , Matsue T. , Hoshi H.
Published: December 2006

Oxygen consumption is a ubiquitous parameter that can provide valuable information on metabolic mechanisms and on oocyte and embryo quality. Recently, we succeeded in non-invasively and quantitatively determining oxygen consumption of individual bovine embryos by scanning electrochemical microscopy (SECM). The aim of this study was to assess by SECM the oxygen consumption of bovine cumulus cells and oocytes cultured in serum-free and serum-supplemented media for oocyte maturation. Bovine cumulus–oocyte complexes (COCs) were obtained from ovarian follicles 2–6 mm in diameter. COCs were cultured in IVMD101 medium for serum-free culture and HPM199 medium supplemented with 5% calf serum (HPM199+CS) for serum-supplemented culture in a humidified atmosphere of 5% CO2 in air (20% O2) at 38.5°C for 24 h. Oxygen consumption by single COCs was non-invasively quantified by a SECM measuring system (Abe et al. 2004 J. Mamm. Ova Res. 21, 22). After the measurements, COCs were treated with 0.5% pronase to completely remove the cumulus cells. The oxygen consumption of single denuded oocyte was measured by SECM. Some COCs and oocytes were prepared for transmission electron microscopy. Oxygen consumption has been monitored in COCs and oocytes cultured in IVMD101 and HPM199+CS media for oocyte maturation (Table 1). Oxygen consumption rates of the immature COCs and denuded oocytes (immediately upon recovery from ovary: control) were 6.91 and 0.70 (×10−14 mol s−1), respectively. In serum-free culture (IVMD101), an increase in oxygen consumption rate was found in oocytes, whereas the oxygen consumption of COCs decreased during oocyte maturation. On the other hand, the oxygen consumption of COCs and oocytes cultured in serum-supplemented medium (HPM199+CS) were not change compared with that of controls. Electron microscopic study demonstrated that the mitochondria moved from a peripheral location in the ooplasm to an even spatial distribution in the oocytes cultured in IVMD101 medium, whereas many of the mitochondria in oocytes cultured in HPM199+CS were distributed in the peripheral region of the ooplasm after oocyte maturation. These results suggest that the respiration activity of bovine cumulus cells and oocytes changed during oocyte maturation, and the respiration activity and ultrastructural features of oocytes may affect the culture conditions. The SECM procedures may provide valuable information on oocyte quality and culture conditions for oocyte maturation.

Table 1.
Oxygen consumption rates (F × 10−14 mol s−1) of the bovine COCs and oocytes in oocyte maturation cultures
320 MPF ACTIVITY AND DEVELOPMENTAL COMPETENCE IN DIFFERENT SIZES OF PREPUBERTAL GOAT OOCYTES
Authors: Anguita B. , Jimenez-Macedo A. R. , Izquierdo D. , Paramio M. T.
Published: December 2006

Developmental competence of prepubertal goat oocytes recovered from a slaughterhouse is low, probably due to an incomplete cytoplasmic maturation. Regulation of cytoplasmic maturation is still unknown, although maturation-promoting factor (MPF) is suggested to play an important role in this process. To better understand the role of MPF in cytoplasmic maturation, we have studied MPF kinase activity in oocytes with different developmental competence. Ovaries were obtained from a slaughterhouse, and oocytes were recovered by slicing and were selected according to morphological criteria. Some oocytes were denuded and classified in diameter groups (<110 μm, 110–125 μm, 125–135 μm, and >135 μm), placed in lysis buffer (50 mM Tris-HCl (pH 7.5), 0.5 M NaCl, 5 mM EDTA, 0.01% Brij35, 1 mM phenyl methyl sultonyl fluoride (PMSF), 0.05 mg/mL leupeptin, 50 mM 2-mercaptoethanol, 25 mM α-glycerophosphate, 1 mM Na-orthovanadate) and frozen in liquid N2. Cell extracts were stored at −80°C until use. The rest of oocytes were matured in vitro in medium TCM199 supplemented with hormones, 10% (DBS), and 400 μM cysteamine, for 27 h in 5% CO2 in air and 38.5°C. After IVM, a sample of oocytes were also denuded, classified by diameters, and frozen as described above. The rest of oocytes were used for IVF in mDM with spermatozoa capacitated with heparin and ionomicin. After 24 h, presumptive zygotes were cultured for 7 days in medium SOF in 5% CO2, 5% O2, and 90% N2 at 38.5°C. At 48 h post-insemination, we added 0.1 μL FBS per embryo. Embryonic development was evaluated with Hoechst staining after IVC. MPF kinase activity was detected using the MESACUP cdc2 kinase assay kit (MBL Woburn, MA, USA). Briefly, the oocyte extract corresponding to 10 oocytes was mixed with 10× reaction buffer (25 mM HEPES buffer (pH 7.5), 10 mM MgCl2) and 10% biotinylated MV peptide (SLYSSPGGAYC). We added 0.1 mM ATP to start the reaction. The mixture was incubated at 30°C for 30 min. The reaction was finished by adding 200 μL of PBS containing 50 mM EGTA. The phosphorylated MV peptide was detected by specific antibody using an ELISA procedure, and the OD was measured at 492 nm. Fisher's exact test was used to analyze IVC results, and ANOVA to analyze cdc2 kinase activity results. We considered differences statistically significant when P < 0.05. Results are shown in Table 1. We observed that embryonic cleavage and blastocyst rate increased with oocyte diameter. The MPF activity detected was higher in the largest oocytes after IVM. As a consequence, we could establish that oocytes with a higher MPF activity are more capable of maintaining embryonic development until the blastocyst stage, which may indicate the important role that MPF plays in cytoplasmic maturation.

Table 1.
Cleavage, blastocyst rate, and MPF kinase activity in different sizes of prepubertal goat oocytes
321 OOCYTE DIAMETER INFLUENCES THE MEIOTIC RESUMPTION AND PROGRESSION INDUCED BY OKADAIC ACID IN DOG
Authors: Ariu F. , Bogliolo L. , Rosati I. , Zedda M. T. , Pau S. , Ledda S.
Published: December 2006

The acquisition of meiotic competence, in the bitch as in many other mammalian species, is related to the oocyte diameter. This study was designed to determine the effect of okadaic acid (OA), a potent inhibitor of seronine/threonine 1 and 2A phosphatases, on meiotic resumption and progression in canine oocytes with different diameters. In two experiments, healthy cumulus-oocytes complexes were collected from ovaries of bitches at various stages of the estrous cycle and divided, by diameters, into three treatment groups for in vitro maturation: <110 �m, 110-120 �m, and >120 �m. In Experiment 1, oocytes were pre-incubated for 1 h in TCM-199 + 20% estrous canine serum (SCE) + cysteamine + OA (0.5 �M). Then, oocytes were cultured for 48 h in the same medium without OA at 38.5�C, 5% CO2 in air. As a control group, oocytes were matured in vitro under the same conditions but without pre-incubation with OA. In Experiment 2, to determine if the effect of OA is mediated by cumulus cells, >120 �m oocytes were denuded from cumulus cells, incubated with or without OA, and cultured in vitro as previously described. At 48 h, all oocytes were stained and fixed with glycerol-Hoechst 33342 to assess the stage of meiotic maturation. In Experiment 1, OA induced a significantly higher incidence of meiotic resumption in oocytes <110 �m (16/108, 14.8%; P < 0.05) and 110-120 �m (70/130, 53.8%; P < 0.01) as compared to that of oocytes in the <110 �m and 110-120 �m control groups (2/58, 3.4%; 24/82, 29.3%). The percentage of oocytes in the 110-120 �m OA group that underwent in vitro maturation to metaphase II (MII) was significantly higher than in the 110-120 �m control group (18/130, 13.8% vs. 4/82, 4.9%, respectively; P < 0.05). In contrast, smaller oocytes (<110 �m) did not develop to MII with or whitout OA. Meiotic resumption rate of >120 �m OA group (64/78, 82.0%) was similar to the >120 �m control group (56/72, 77.8%), but a significantly higher proportion of the oocytes pre-incubated with OA progressed to MII than did the control oocytes (40/78, 51.3% vs. 12/72, 16.7%, respectively; P < 0.01). Low rates of meiotic resumption were observed in denuded >120-�m oocytes with (7/63, 11.1%) or without OA (7/55, 12.7%) and none of them progressed to MII. In conclusion, the results of the present study indicate that treatment of fully grown (>120 �m) oocytes with okadaic acid at the onset of in vitro maturation can result in a higher frequency of meiotic maturation than previously reported. Also, we determined that the beneficial effect of okadaic acid was mediated by cumulus cells.
322 LEPTIN TREATMENT DURING BOVINE OOCYTE MATURATION AFFECTS mRNA LEVELS OF APOPTOSIS-RELATED GENES
Authors: Boelhauve M. , Paula-Lopes F. F. , Habermann F. A. , Sinowatz F. , Wolf E.
Published: December 2006

The series of events associated with oocyte growth and maturation determines its ability to undergo successful fertilization, cleavage, and embryonic development. Among the molecules involved in these events, leptin has been identified as a modulator of oocyte function. The latest studies have focused on long-term effects of leptin during maturation of bovine oocytes on apoptosis and gene expression in in vitro-produced blastocysts. Briefly, blastocysts originating from leptin-treated oocytes exhibited decreased transcript levels of BCL2 associated X-protein (BAX), but increased mRNA concentrations for leptin receptor (LEPR), signal transducer and activator of transcription 3 (STAT3), and baculoviral inhibitor of apoptosis protein repeat-containing 4 (BIRC4, also known as XIAP) (Boelhauve et al. 2005 Biol. Reprod. 73, 737-744). In the present study, we analyzed single oocytes and their surrounding cumulus cells matured in the presence of 0, 1, and 10 ng/mL leptin. Transcript levels of LEPR, STAT3, BAX, BIRC4, FASLG (encoding Fas ligand), and FAS (encoding Fas receptor) were determined by reverse transcriptase-quantitative PCR (RT-qPCR) analysis of matured oocytes and cumulus cells. Four IVM replicates with four individual samples were collected 22 h after the start of IVM. Total RNA was isolated using a modified TriZol protocol, and reverse transcribed using the enzyme Superscript II-RT and random hexamer primers. Quantitative PCR was conducted with SYBR-Green as a double-stranded DNA-specific fluorescent dye in an ABI 7000 SDS apparatus. Treatment of COCs with 1 or 10 ng/mL leptin increased transcript levels of LEPR (2-fold; P < 0.001), STAT3 (2-fold; P < 0.001), BAX (2-fold; P < 0.001) and FAS (2-fold; P < 0.001) in cumulus cells. Interestingly, the transcript level of the well-known inhibitor of apoptosis, BIRC4, was increased about 4-fold (P < 0.001). In oocytes, leptin treatment increased the mRNA abundance of STAT3 (10 ng/mL; P < 0.05), FAS (1 ng/mL; P < 0.05 and 10 ng/mL; P < 0.001), and FASLG (10 ng/mL; P < 0.05). In conclusion, physiological doses of leptin during maturation of COCs have effects on the expression of developmentally important and apoptosis-associated genes in the oocyte and surrounding cumulus cells. The higher level of BIRC4 mRNA in leptin-treated cumulus cells was associated with a reduced proportion of cumulus cell apoptosis, which might explain the positive and long-lasting effects of leptin on the developmental competence of bovine oocytes.
325 ACTIVITY OF MATURATION-PROMOTING FACTOR (MPF) AND MITOGEN-ACTIVATED PROTEIN KINASES DURING IN VITRO MATURATION OF BUFFALO OOCYTES (BUBALUS BUBALIS)
Authors: Gasparrini B. , Leoni G. , Boccia L. , Galiotto M. , Ledda S. , Zicarelli L.
Published: December 2006

The maturation promoting factor (MPF) and mitogen-activated protein kinases (MAPK) are the key regulators of both meiotic and mitotic cell cycles. The absence of data on the activity of the major cell cycle kinases in buffalo oocytes during meiotic progression provided the bases for this study. More specifically we assayed the MPF and MAP kinase activity of buffalo oocytes during meiosis. Abattoir-derived cumulus-oocyte complexes (COCs) with a compact, non-atretic cumulus and a homogeneous cytoplasm were utilized for the study. The COCs (n = 293, over four replicates) were matured in vitro in TCM-199 supplemented with 10% fetal calf serum (FCS), 0.2 mM sodium pyruvate, 0.5 �g/mL FSH, 5 �g/mL LH, 1 �g/mL 17�-estradiol, 50 �M of cysteamine, and 50 �g/mL kanamycin (B199). In vitro maturation (IVM) was carried out at 38.5�C under a controlled gas atmosphere of 5% CO2 in humidified air. At scale times during the culture (0, 3, 6, 9, 12, 15, 18, 21, 24 h) groups of oocytes were stained with Hoechst 33342 to assess chromatin configuration and stored according to the maturation stage (GV, GVDB, MI, and MII) at -80�C pending protein analysis. SDS-polyacrylamide gel electrophoresis wase performed using Laemmli discontinuous buffer system (Laemmli 1970 Nature 227, 680) with a 12% running gel. Groups of oocytes were analyzed for MPF activity (n = 65) by histone H1 kinase activity (Naito and Toyoda 1991 J. Reprod. Fertil. 93, 467-473) and for MAPK activity (n = 48) by myelin basic protein assays (Chesnel et al. 1995 Biol. Reprod. 52, 895-902). The activity of both MPF and MAP kinases was quantified by measuring the density of the bands on the autoradiographic film with a densitometer. Differences in the levels of the kinases among groups were analyzed by ANOVA. It was assumed that the value of MPF and MAPK was 100% in metaphase II (MII) stage oocytes. The lowest levels of MPF and MAPK activities were found in the oocytes at the GV (0-6 h post-IVM: 40% and 17.2%, respectively) and at the GVBD (6-9 h post-IVM: 41.2% and 18%) stages. The activities increased at metaphase I (MI) stage (9-15 h post-IVM) and at MII (21-24 post-IVM). Interestingly, although similar levels of MAP kinases were found at MI and MII stages (95.1% vs. 100%), MPF levels were significantly lower (P < 0.01) at the MI stage compared to those detected at MII (82.8% vs. 100%). The fluctuations of the MPF levels in buffalo appear different compared to those observed in other species; in particular, no differences were recorded between the GV and the GVBD stages whereas a significant increase of the MPF levels was found at MII compared to the MI stage. It seems that MPF and MAPK could differently guide meiotic resumption and progression to the MII arrest in this species. To our knowledge, this is the first report on biochemical analysis of the cell cycle regulation in buffalo oocytes.
328 EPIDERMAL GROWTH FACTOR ENHANCES MEIOTIC MATURATION OF CANINE OOCYTES IN THE PRESENCE OF BSA
Authors: Jin Y.-X. , Shen X.-H. , Cui X.-S. , Kong I.-K. , Kim N.-H.
Published: December 2006

Despite many attempts to improve the in vitro maturation (IVM) of canine oocytes using various culture conditions, the efficiency of canine IVM remains very low compared with that of other domestic animals. In the present study we examined the effect of (1) ovarian estrous stage on oocyte quality, and (2) epidermal growth factor (EGF) in the presence and absence of macromolecules on the IVM of canine oocytes. Reproductive tracts from normal, client-owned bitches aged approximately 7 months were collected after routine ovariohysterectomy at private veterinary clinics. The ovaries were removed, washed free of blood with PBS, and repeatedly cut with a scalpel blade at room temperature in HEPES-buffered Tyrodes medium containing 0.1% (w/v) polyvinyl alcohol. Each group of 10 cumulus-oocyte complexes (COCs) was matured in North Carolina State University (NCSU)-17 medium supplemented with 1 �g/mL FSH, 10 IU/mL hCG, and 1 �g/mL �-estradiol under paraffin oil for 48 or 72 h at 37�C. Follicular ovaries contained the largest number of oocytes (more than 300), and the number of oocytes e100 �m in diameter was also significantly greater in follicular ovaries than in ovaries at other stages (P < 0.001). Luteal ovaries had the smallest number of both total and large oocytes. After 72 h of culture, significantly more oocytes recovered from follicular ovaries were in GVBD than oocytes from anestrous or luteal ovaries. After 72 h of culture, oocytes recovered from follicular ovaries had a significantly lower percentage reaching the GV stage (59.7 � 5.2%) and more in GVBD (40.3 � 3.1%) compared with both anestrous (GV, 75.1 � 5.2%; GVBD, 24.9 � 3.1%) and luteal (GV, 79.8 � 5.2%; GVBD, 22.0 � 3.1%) ovaries (P < 0.05). However, maturation to MI/MII was not statistically different between the stages of the estrous cycle. BSA or FBS supplementation did not improve meiotic maturation when compared to PVA supplementation. In the presence of 100 nM epidermal growth factor (EGF), either FBS or BSA enhanced the proportion of oocytes in GVBD/MI (59.7 � 2.2% [FBS] and 51.7 � 2.2% [BSA] vs. 36.1 � 2.2% [PVA], P < 0.05). In addition, 0.4% BSA significantly increased the percentage of oocytes in MII (18.5 � 0.8%) compared to 10% FBS (7.1 � 0.8%; P < 0.05). These results suggest that the estrous cycle of bitches influences the meiotic maturation of oocytes cultured in vitro, and EGF increases the meiotic maturation of canine oocytes in the presence of BSA in vitro.
This work was funded by a grant from the Ministry of Science and Technology of Korea and the National Research Laboratory Program.