Differences In Cleavage Rates Research Articles

37 THE EFFECTS OF A CLASS III HISTONE DEACETYLASE INHIBITOR (SIRTINOL) ON EARLY DEVELOPMENT OF PORCINE CLONED EMBRYOS

Previous studies have demonstrated that treatment of cloned embryos with trichostatin A (TSA) or scriptaid, inhibitors of class I and II histone deacetylases (HDAC), significantly enhanced their developmental competence. In the present study, we investigated the effects of sirtinol, an inhibitor of class III HDAC, on the embryonic development of porcine cloned embryos. Data were analyzed with SPSS 17.0 software (SPSS Inc., Chicago, IL, USA) using Duncan’s multiple range test and all experiments were replicated at least 5 times. In experiment 1, 648 parthenotes were divided into 4 groups (0-, 6-, 12-, and 24-h sirtinol treatment after activation) to investigate optimal treatment time using 100 µM sirtinol. The cleavage rate of the 24-h treatment group (81.3%) was significantly (P < 0.05) decreased compared with the 12-h treatment group (88.4%) but there was no difference compared with the control (86.9%) and 6-h treatment groups (86.9%). The parthenotes treated with sirtinol for 12 h after activation had a significantly higher blastocyst formation rate and total cell number in blastocysts (50.5% and 66.9, respectively) than the control (39.4% and 54.1, respectively). In experiment 2, 806 cloned embryos were divided into 5 groups (0, 50, 100, 150, and 200 µM sirtinol treatment for 12 h after activation) to investigate optimal concentration. There was no significant difference in cleavage rate. The rate of blastocyst formation and total cell number in blastocysts were significantly (P < 0.05) improved by treatment with 150 µM sirtinol for 12 h after activation (28.8% and 51.0, respectively) compared with the control (17.5% and 37.1, respectively). The total cell number in blastocysts was also significantly increased in 50 and 200 µM groups (47.9 and 48.4, respectively) compared with the control (37.1). In experiment 3, we examined the effects of 150 µM sirtinol treatment for 12 h after activation with or without 5 nM TSA on in vitro embryonic development after somatic cell nuclear transfer. The rate of blastocyst formation was significantly improved in sirtinol-treated and TSA-treated groups (30.9 and 31.3%, respectively) but not in the sirtinol with TSA group (27.6%) compared with the control (21.7%). The total cell number in blastocysts was significantly increased by treatment with sirtinol and TSA together (73.9) compared with the control (49.0) but there was no difference in only sirtinol- (59.8) or TSA- (59.2) treated groups. There was no significant difference in cleavage rate among groups. Our results suggest that sirtinol improves the embryonic development of porcine cloned embryos and sirtinol with TSA synergistically increases the blastocyst quality. This work was supported by a grant from the Next-Generation BioGreen 21 program (no. PJ008121012011), Rural Development Administration, Republic of Korea.

The in vitro and in vivo developmental capacity of selected porcine monospermic zygotes

In this study, we evaluated the in vitro and in vivo developmental capacity of selected monospermic zygotes produced in vitro. Cumulus-oocyte complexes were matured in vitro and inseminated with frozen-thawed spermatozoa. Thirteen hours after insemination, presumptive zygotes were centrifuged at 15,000 ×g for 20 minutes to polarize the lipids in the cytoplasm and permit the visualization of pronuclei. Then, the oocytes were individually classified as bipronuclear (2PN) or polypronuclear (three or more pronuclei, PPN). To examine embryo development, 102 selected zygotes were cultured for 7 days. There were no differences in cleavage rate (93.0% and 88.9% for 2PN and PPN zygotes, respectively). However, the blastocyst formation rate was higher (P < 0.003) in 2PN (80.7%) zygotes than in PPN (53.3%) zygotes. The control (noncentrifuged, nonselected zygotes) group showed lower (P < 0.003) cleavage rate and blastocyst formation than the 2PN and PPN zygotes. In a second experiment, 2PN zygotes and control zygotes were transferred (30 zygotes per transfer) by laparoscopy into the oviducts of recipient gilts (10 recipients per group) on the first day of standing estrus. The farrowing rates were 70% and 40% for transfers made with 2PN and control zygotes, respectively. The average number of piglets born per recipient farrowed did not differ between groups (4.9 ± 0.6 and 4.5 ± 1.2, respectively), but the efficiency (number of live piglets per total transferred embryos) was higher (P < 0.01) for 2PN zygotes than for the control group (9.3% and 4.0%, respectively). These results demonstrate the effectiveness of centrifugation for the selection of monospermic zygotes as a procedure to improve in vitro embryo production in pigs. In addition, the results indicate that the laparoscopic technique described here is a simple and effective procedure for transferring embryos into one oviduct.

Expression patterns of sirtuin genes in porcine preimplantation embryos and effects of sirtuin inhibitors on in vitro embryonic development after parthenogenetic activation and in vitro fertilization

We examined the expression patterns of porcine sirtuin 1 to 3 (Sirt1–3) genes in preimplantation embryos derived from parthenogenetic activation (PA), in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT). We also investigated the effects of sirtuin inhibitors (5 mM nicotinamide [NAM] and 100 μM sirtinol) on embryonic development of PA and IVF embryos under in vitro culture (IVC). The expression patterns of Sirt1–3 mRNA in preimplantation embryos of PA, IVF, and SCNT were significantly (P < 0.05) decreased from metaphase stage of oocyte to blastocyst stage. Especially, the expressions of Sirt1–3 in SCNT blastocysts were significantly (P < 0.05) lower and Sirt2 in PA blastocyst was significantly higher compared with the IVF blastocysts. Treatment with sirtuin inhibitors during IVC resulted in significantly (P < 0.05) decreased blastocyst formation and total cell number of blastocyst derived from PA (NAM: 29.4% and 29.6, sirtinol: 31.0% and 30.3, and control: 40.9% and 41.7, respectively) and IVF embryos (NAM: 10.4% and 30.9, sirtinol: 6.3% and 30.5, and control: 16.7% and 42.8, respectively). There was no significant difference in cleavage rate in both PA and IVF embryos. The early and expanded blastocyst formations at Day 7 were significantly lower in the sirtuin inhibitors-treated groups than the control. It was demonstrated that sirtuin inhibitor (NAM) influenced the percentage of blastocyst formation and total cell number of PA derived blastocyst when NAM was added during Day 4 to 7 (22.1% and 32.4) or Day 0 to 7 (23.1% and 31.6) of IVC compared with the control (41.8% and 41.5). No significant difference in cleavage rates appeared among the groups. The blastocysts derived from PA embryos treated with sirtuin inhibitors showed lower (P < 0.05) expressions of POU5F1 and Cdx2 genes. Also, Sirt2 mRNA expression was significantly decreased in sirtinol treated group and Sirt3 mRNA expression was also significantly decreased in both NAM and sirtinol treated groups compared with the control. In conclusion, these results suggest that sirtuins may have a physiological and important role in embryonic development of porcine preimplantation embryos by regulating essential gene expressions of developing embryos. These findings could have implications for understanding the role of sirtuins during embryo development and for improving SCNT and related techniques.

Porcine androgenetic embryos develop to fetal stage in recipient mothers

In livestock, parthenogenic embryos are simple to produce, but androgenetic embryos have been successfully produced only in sheep and cows. In the present study, matured porcine oocytes were enucleated by micromanipulation and then fertilized with sperm in vitro, thereby producing porcine androgenetic embryos. Porcine androgenetic embryos, which had only sperm genomes, were assessed for cleavage and for blastocyst formation 2 and 6 d after IVF, respectively. There was no difference in cleavage rate between androgenetic embryos and biparental IVF embryos (mean ± SD androgenetic: 65.5 ± 5.4%; biparental IVF: 63.2 ± 3.6%), but there was a difference in the rate of blastocyst formation (androgenetic: 4.5 ± 0.7%; biparental IVF: 30.2 ± 2.6%, P < 0.05). The average number of cells in Day 6 androgenetic blastocysts (34.3 ± 18.2) was lower (P < 0.05) than that in biparental IVF blastocysts (44.1 ± 19.5), but did not differ from that in parthenogenetic embryos (35.7 ± 16.7). The androgenetic embryos were transferred into recipient mothers to examine the competence of post-implantation development. Androgenetic fetuses were present on Days 21 and 25, but not on Days 28, 31, or 35. Of the six androgenetic fetuses recovered on Day 21, five had normal, translucent bodies, and two of these five had beating hearts. The four fetuses recovered on Day 25 were all non-viable. In conclusion, porcine androgenetic embryos initiated embryogenesis and had reached a viable fetal stage 21 days after IVF.

189 THE EFFECTS OF RESVERATROL ON PORCINE OOCYTES IN VITRO MATURATION AND SUBSEQUENT DEVELOPMENT AFTER PARTHENOGENETIC ACTIVATION AND IN VITRO FERTILIZATION

The present study investigated the effects of resveratrol (a phytoalexin with various pharmacological activities) during in vitro maturation (IVM) of porcine oocytes on nuclear maturation, intracellular glutathione (GSH) and reactive oxygen species (ROS) levels, gene expression in matured oocytes and subsequent embryonic development after parthenogenetic activation (PA) and IVF. Data were analyzed with SPSS 17.0 using Duncan's multiple range test. In experiment 1, a total of 1146 cumulus–oocyte complexes (COC) were divided into 5 groups (0, 0.1, 0.5, 2.0 and 10.0 μM resveratrol). In the nuclear maturation after 44-h IVM, the groups of 0.1, 0.5 and 2.0 μM (83.0, 84.1 and 88.3%, respectively) had no significant difference compared to the control group (84.1%). The group of 10.0 μM decreased the nuclear maturation (75.0%) significantly (P &lt; 0.05). In experiment 2, a total of 300 matured oocytes were examined for the effects of different resveratrol concentrations (0, 0.5, 2.0 and 10.0 μM) on porcine oocyte intracellular GSH and ROS levels. The groups of 0.5 and 2.0 μM showed a significant (P &lt; 0.05) increase in intracellular GSH levels (1.3 and 1.3, respectively) compared with the control and 10.0 μM groups (1.0 and 1.0, respectively). The intracellular ROS level of oocytes matured with 2.0 μM resveratrol (0.4) was significantly (P &lt; 0.05) decreased compared to other groups (control: 1.0; 0.5 μM: 0.6; and 10.0 μM: 0.7). In experiment 3, lower expression of apoptosis-related genes (Bax, Caspase-3 and Bak) was observed in matured oocytes treated with 2.0 μM resveratrol when compared with that of the control (P &lt; 0.05). In experiment 4, a total of 728 oocytes were divided into 4 groups (control, 0.5, 2.0 and 10.0 μM) and examined subsequent to embryonic development after PA. Oocytes treated with 2.0 μM resveratrol during IVM had a significantly higher cleavage (CL) rate, blastocyst (BL) formation rate and total cell numbers (TCN) after PA compared with those of the control (2.0 μM: 96.6%, 62.1% and 49.1 vs control: 88.3%, 48.8% and 41.4, respectively) and the 10.0 μM groups (87.3%, 41.4% and 40.9, respectively). Oocytes treated with 0.5 μM resveratrol (87.2%, 50.5% and 48.6, respectively) during IVM had significantly higher TCN, but there were no differences in CL and BL formation rates. In experiment 5, a total of 935 oocytes in 3 groups (control, 2.0 and 10.0 μM resveratrol) were conducted in IVF. The BL formation rate and TCN were significantly higher in the group of 2.0 μM resveratrol (20.5% and 54.0, respectively) than the control (11.0% and 43.4, respectively) and 10.0 μM group (11.7% and 45.0, respectively), but there was no significant difference in CL rate. In conclusion, 2.0 μM resveratrol supplementation during IVM improved the developmental potential of PA and IVF in porcine embryos by increasing the intracellular GSH concentration, decreasing the ROS level and decreasing apoptosis-related gene expression during oocyte maturation. This work was supported by a grant from the Next-Generation BioGreen 21 Program (No. PJ008121), Rural Development Administration, Republic of Korea.

Effects of porcine granulocyte-macrophage colony-stimulating factor on porcine in vitro-fertilized embryos

This study investigated the effects of porcine granulocyte-macrophage colony-stimulating factor (pGM-CSF) on the developmental potential of porcine in vitro-fertilized (IVF) embryos in chemically and semidefined (with BSA) medium. In experiment 1, zygotes were treated with different concentrations of pGM-CSF (0, 2, 10, 100 ng/mL). The results indicated that 10 ng/mL pGM-CSF significantly (P < 0.05) increased blastocyst development and total cell number (15.1% and 53.5, respectively) compared with the control (6.1%, and 38.8, respectively). Comparing blastocyst formation, early and expanded blastocyst formation was significantly higher in the 10 ng/mL-pGM-CSF group than in the control on Days 6 and 7 of the culture period. However, there was no significant difference in cleavage rate. Experiment 2 demonstrated that pGM-CSF influenced the percentage of blastocyst formation and total cell number when pGM-CSF was added during Days 4 to 7 (14.6% and 53.9, respectively) or Days 0 to 7 (15.2% and 54.0, respectively) compared with the control (7.8% and 43.1, respectively) and compared with Days 0 to 3 (8.7% and 42.5, respectively). Similarly, early blastocyst formation rates were significantly higher at Days 4 to 7 than in the control, and expanded blastocyst formation was significantly higher at Days 4 to 7 or Days 0 to 7. No significant difference in cleavage rates appeared among the groups. In experiment 3, in the presence of BSA, pGM-CSF also increased the percentage of embryos that developed to the blastocyst stage and the total cell number (20.3% and 59.8, respectively) compared with the control (14.9% and 51.4, respectively), whereas there was no significant difference in cleavage rate. Experiment 4 found that the total cell number and the number of cells in the inner cell mass (ICM) were significantly increased compared with the control when zygotes were cultured in either porcine zygotic medium (PZM)-3 or PZM-4 supplemented with 10 ng/mL pGM-CSF. The number of trophectoderm (TE) cells was significantly higher in PZM-3 medium supplemented with pGM-CSF than in the control, and the number tended to increase (P = 0.058) in PZM-4 medium supplemented with pGM-CSF. The ratio of inner cell mass to trophectoderm cells was significantly higher in PZM-4 supplemented with 10 ng/mL pGM-CSF, but not in PZM-3. In experiment 5, it was found that the male pronuclear formation rate, monospermic penetration and sperm/oocyte were 95.4%, 37.2%, and 2.4, respectively. Together, these results suggest that pGM-CSF may have a physiological role in promoting the development of porcine preimplantation embryos and regulating cell viability and that addition of pGM-CSF to IVC medium at Days 4 to 7 or 0 to 7 improves the developmental potential of porcine IVF embryos.

190 EFFECT OF TRANS-ε-VINIFERIN ON IN VITRO PORCINE OOCYTE MATURATION AND SUBSEQUENT DEVELOPMENTAL COMPETENCE IN PRE-IMPLANTATION EMBRYOS

Trans-ε-viniferin is a naturally occurring polyphenol belonging to the stilbenoids family. Trans-ε-viniferin is isolated from Vitis amurensis, 1 of the most common wild grapes in Korea, Japan and China. We investigated the effects of trans-ε-viniferin on in vitro maturation (IVM) and developmental competence after IVF or parthenogenesis (PA). At the laboratory of Veterinary Pharmacology, College of Veterinary Medicine, Chungbuk National University, trans-ε-viniferin was purified from the leaves and stems of Vitis amurensis. Data were analyzed with SPSS 17.0 using Duncan's multiple range test. First, in total, 594 cumulus–oocyte complexes (COC) were used for the evaluation of nuclear maturation. The COC were matured in TCM-199 medium supplemented with various concentrations of trans-ε-viniferin (0, 0.1, 0.5, 1.0 and 5.0 μM) with 10% porcine follicular fluid, 10 IU mL–1 of eCG and 10 IU mL–1 of hCG. After 22 h in maturation culture, the COC were cultured in hormone-free medium supplemented with various concentrations of trans-ε-viniferin for an additional 22 h and then nuclear maturation was evaluated. Second, in total, 300 matured oocytes were used to examine the effects of different trans-ε-viniferin concentrations (0, 0.5 and 5.0 μM) on porcine oocyte intracellular glutathione (GSH) and reactive oxygen species (ROS) levels. Lastly, the developmental competence of oocytes matured with different concentrations of trans-ε-viniferin (0, 0.5 and 5.0 μM) was evaluated after IVF or PA. In total, 711 embryos were evaluated. As results, we observed that trans-ε-viniferin treatment during IVM did not improve the nuclear maturation of oocytes in any group (84.2, 86.6, 85.5, 83.3 and 79.2%, respectively), but significantly increased (P &lt; 0.05) intracellular GSH levels in the 0.5 μM group (0 μM vs 0.5 μM; 14.6 vs 16.8 pmol oocyte–1) and reduced ROS levels (0 μM vs 0.5 μM and 50 μM; 174.6 vs 25.7 and 23.8 pixel oocyte–1). Oocytes treated with trans-ε-viniferin during IVM did not have significantly different cleavage rates or blastocyst formation rates after IVF, but total cell numbers were significantly higher (P &lt; 0.05) in the 0.5 and 5.0 μM treatment groups (53.6 ± 4.0 and 47.9 ± 3.1) compared to the control group (36.4 ± 2.2). The PA embryos showed similar results; there were no significant differences in cleavage rates and blastocyst formation rates, but the total cell number significantly increased in the 0.5 and 5.0 μM treatment groups (59.6 ± 4.2 and 60.8 ± 4.6) compared to the control group (43.1 ± 2.1). In conclusion, these results indicate that trans-ε-viniferin treatment during porcine IVM increased total cell number of blastocysts, possibly through increasing intracellular GSH synthesis and reducing ROS levels. This work was supported by a grant from the Korea institute of Planning &amp; Evaluation for Technology in Food, Agriculture, Forestry &amp; Fisheries, Republic of Korea.

Improved Developmental Competence and Spindle Assembly of In Vitro Maturation of Human Oocytes by Prolonged Culture Duration.

In-vitro maturation of human oocytes is a promising technology for the treatment of human infertility, however it is still unclear that how to obtain the similar development competence of matured oocytes during the period of oocyte maturation compared with fresh MII oocytes. The purpose of this study was to investigate the relation between prolong culture time after maturation and subsequent embryo development results, and develop an in vitro oocyte maturation (IVM) method for a wide range of patients. Immature oocytes (including MI and GV oocytes) that underwent meiotic maturation during brief in vitro culture and they were subjected to parthenogenesis using artificial activation method through different prolonged culture duration. Morphology of oocytes and 8-cell embryos, fertilization, cleavage and blastocyst development was evaluated in each group. A total of 486 immature oocytes were collected in 122 IVM-ICSI cycles. Oocyte was separated into four groups with different prolonged culture duration. The overall activation rate is significant lower in both 0- and 4-hour groups compared with other two groups, and there is no difference in cleavage rate in all groups. In the stage of zygotic genomic activation, there is no difference among 0-, 2- and 4-hour groups, but significant higher than 8-hour group. In both 2- and 4-hour group, the efficiency of blastocyst formation is significant higher than that of other two groups. The Grading results at 8-cell stage showed that embryos with high quality was exceeded to 50 percent in 0-, 2- and 4-hour groups, whereas this proportion is less than 30 percent in 8-hour group. Immunostaning results demonstrated that the spindle is not separated completely between nuclear and polar body in 0-hour group, and the spindle lost partial or total disorganization or complete lack of microtubules, and the chromosomes began to condense in 8-hour group. This present study concluded that correctly prolonged culture duration will benefit for the development competence of oocytes matured in vitro, and facilitated the application of immature oocytes in clinical. (poster)

Effects of chemically defined medium on early development of porcine embryos derived from parthenogenetic activation and cloning

The present study was to investigate if a completely chemically defined medium (PZM-4) could support the early development of porcine embryos derived from parthenogenetic activation (PA) and cloning (somatic cell nuclear transfer, SCNT), and to lay the foundation for determining the physiological roles of certain supplements in this medium. Porcine embryos derived from PA and SCNT were cultured in media: PZM-3 (a chemically semi-defined medium), PZM-4 (a fully defined medium), and PZM-5 (an undefined medium). Early embryo development was observed. We found that the three medium groups (PZM-3, PZM-4 and PZM-5) exhibited no significant differences in cleavage rates of PA embryos (p > 0.05), while the blastocyst rate in PZM-3 was significantly higher than in PZM-4 and PZM-5 (78.9% vs. 36.0% and 52.3%) (p < 0.05). Moreover, total cell number per blastocyst in PZM-3 was clearly higher than in PZM-5 but similar to that in PZM-4. As for SCNT embryos, no significant differences were observed for the cleavage rates or the blastocyst rates among the three groups (p > 0.05). However, total cell number per blastocyst in PZM-3 was notably higher than in PZM-5, but was similar to that in PZM-4. In conclusion, our results suggested that the completely chemically defined medium PZM-4 can be used to efficiently support the early development of porcine PA and SCNT embryos.

139 TRICHOSTATIN A IMPROVED IN VITRO DEVELOPMENT OF PORCINE NUCLEAR TRANSFER EMBRYOS PRODUCED BY A NEW ACTIVATION METHOD

Recently, it has been reported that a new activation method, brief exposure to cycloheximide before electrical activation, could increase development rates and reduce cell death. In our study, we allocated reconstructed SCNT embryos into 3 groups: electrical activation followed by exposure to cycloheximide (10 μg mL–1) for 4 h (ELE+CHX); exposure to cycloheximide (10 μg mL–1) for 10 min followed by electrical activation (CHX+ELE); and electrical pulse treatment alone (ELE). We found the CHX+ELE (10 min) group had a similar blastocyst formation rate and total blastocyst number with the ELE+CHX (4 h) group, and both groups could increase in vitro development compared with the ELE group. Trichostatin A (TSA), an inhibitor of histone deacetylase, has been reported to potentially enhance cloning efficiency. We examine the effect of TSA on nuclear transfer embryos produced by the CHX+ELE activation method. The reconstructed embryos were treated with 50 nM TSA for 0 and 36 h. We found that 50 nM TSA for 36 h after activation had an increased blastocyst rate compared with the control (15.35 v. 8.84%; P &lt; 0.05), but there was no difference in cleavage rate or in total blastocyst numbers. Our data demonstrate that TSA treatment could significantly improve pig nuclear transfer embryos produced by a new activation method. S.L.C., W.W. equal contribution; Corresponding author ZH. X.R., ZH. Y.H.; Supported by NSFC (30700574), 863 (2008AA101003).

216 PRONUCLEAR FORMATION AND DEVELOPMENTAL COMPETENCE OF MATURE PORCINE OOCYTES DERIVED FROM SMALL- AND MEDIUM-SIZED FOLLICLES

The maturation rate of oocytes derived from small follicles (SF) is known to be lower than that of oocytes from medium follicles (MF). The objective of this study was to assess the fertilizability and developmental competence of mature SF oocytes that were selected by the presence of the first polar body. Cumulus–oocyte complexes (COC) were aspirated from SF (1 to 2 mm in diameter) or MF (3 to 6 mm in diameter) of prepuberal ovaries. The COC were cultured in modified porcine oocyte medium supplemented with gonadotropins and dibutyryl cAMP for the first 20-h period and then in gonadotropin-free and dibutyryl cAMP-free porcine oocyte medium for another 24 h. Following IVM culture, mature oocytes with the first polar body were selected under a stereomicroscope, co-incubated with spermatozoa in a drop of modified TCM-199 containing 0.4% BSA and 5 mM caffeine for 6 h, and then incubated in porcine zygote medium-5 for 7 days. Sperm penetration, cleavage, and early development of the oocytes were examined before culture in porcine zygote medium-5 on Days 2 and 7 of culture. To analyse the fertilizability and developmental competence of oocytes from the SF and MF groups, sperm penetration, pronuclear formation, cleavage, blastocyst formation, and mean cell number in a blastocyst (as determined by fluorescence observation following Hoechst 33342 staining) were examined. Statistical analysis was performed by ANOVA with a Bonferroni-Dunn post-hoc test (P &lt; 0.05). The percentages of oocytes in which the first polar body could be observed were 51.0 ± 4.5% and 78.5 ± 2.8% for SF- and MF-oocytes, respectively, whereas the maturation rates were 83.8 ± 4.0% and 62.8 ± 4.4% following fixation and staining. When only mature oocytes were co-cultured with sperm for 6 and 9 h, sperm penetration, monospermic penetration, and pronuclear formation were not different (P &gt; 0.33) between mature SF- and MF-oocytes. Although there was no difference in cleavage rates between the mature SF- and MF-oocyte groups, blastocyst formation rate and mean cell number in the blastocyst were higher in mature MF-oocytes (31.0 ± 3.6% and 38.7 ± 1.9 cells, respectively) than in mature SF-oocytes (14.7 ± 3.2% and 31.2 ± 2.0 cells). From these results, we conclude that mature oocytes derived from SF have a similar fertilizability when compared with mature MF-oocytes, but the developmental competence to the blastocyst stage following IVF is significantly lower in mature SF-oocytes than in mature MF-oocytes.

Selection of viable spermatozoa from testicular biopsies: a comparative study between pentoxifylline and hypoosmotic swelling test

To compare fertilization, cleavage, and pregnancy outcome using pentoxifylline and a hypoosmotic swelling (HOS) test to select viable spermatozoa from testicular biopsy specimens. Open, comparative, prospective study. G.S. Medical College and Fertility clinic, Mumbai, India. A total of 50 couples enrolled for infertility treatment having a male factor indication of nonobstructive azoospermia. Assessment of viable spermatozoa using pentoxifylline and using an HOS test from a population of nonmotile spermatozoa obtained from testicular biopsies. Comparison of fertilization, cleavage, and clinical pregnancy rates using viable sperms recovered using pentoxifylline and an HOS test. Viable spermatozoa were obtained in both the study groups. Significantly higher fertilization rates (pentoxifylline 62.05% vs. HOS 41.07%) and clinical pregnancy rates (pentoxifylline 32% vs. HOS 16%) were observed. There was no significant difference in cleavage rates among both groups. We found that obtaining viable spermatozoa using pentoxifylline was more effective in terms of fertilization and pregnancies than obtaining it through an HOS test.

Short-term treatment with 6-DMAP and demecolcine improves developmental competence of electrically or Thi/DTT-activated porcine parthenogenetic embryos

Treatment with 6-dimethylaminopurine (6-DMAP) or demecolcine (DE) for several (at least 2) hours after artificial activation is known to improve in vitro development of porcine embryos. However, several reports have also shown that treatments with these chemicals induce apoptosis. The aim of this study was to find out whether short-term treatment with 6-DMAP and DE combined with electrical or thimerosal/dithiothreitol (Thi/DTT) activation had a beneficial effect on development of parthenogenetically activated porcine oocytes. We additionally treated embryos with 6-DMAP (2 mM) and/or DE (0.4 microg/ml) for a short time (40 min) after an electrical pulse (EP) or Thi/DTT. As a result, short-term treatment with 6-DMAP and DE successfully induced development of electrically or Thi/DTT-activated porcine parthenogenetic embryos with no significant difference in cleavage rate, blastocyst formation rate and total cell number compared with long-term treatment. To find optimal activation protocol, cleavage rate, blastocyst formation rate and total cell number were compared between EP and Thi/DTT treatments. Thi/DTT + 6-DMAP + DE showed significantly higher blastocyst formation rate (36.1 ± 3.5%) and total cell number (46.9 ± 1.0) than other groups (EP + 6-DMAP + DE, EP + Thi/DTT + 6-DMAP + DE: 23.3 ± 3.0%, 42.2 ± 1.1 and 17.2 ± 2.7%, 36.7 ± 1.5, respectively). In conclusion, this study demonstrates that short-term treatment with 6-DMAP and DE is as effective as the standard long-term treatment and Thi/DTT + 6-DMAP + DE exerts a synergistic effect.

Modifications of chemically induced-enucleated nuclear transfer technique by reverse-order nuclear transfer in mouse

To improve the developmental potential of somatic cell cloned embryos derived from demecolcine (DC) induced-enucleated nuclear transfer (INT), we modified the INT procedures by transferring donor nuclei into recipient cytoplasts prior to the induced enucleation of the recipient cytoplasts, and we called this modified INT technique as reverse-order and induced-enucleated nuclear transfer (RINT). Standard nuclear transfer (SNT) and INT were performed as controls. The dynamic changes of maternal and transferred donor nuclei in the RINT oocytes were monitored to evaluate the feasibility of this new nuclear transfer (NT) technique by timed immunofluorescence. Timed immunofluorescence showed that RINT is feasible because none of the transferred donor nuclei were expelled with the second polar body (Pb) in the RINT oocytes, while 42.2% of the oocytes showed extrusion of all maternal chromosome and spindles with the second Pb at 60 min after activation and DC treatment. Although there was no difference in cleavage rate (86.6% vs. 82.1%), the rates of successful enucleation and blastocyst formation were significantly increased in RINT compared with INT (44.1% vs. 27.5% and 43.3% vs. 12.8%, respectively; p < 0.01). Compared with SNT, there was no difference in cleavage rate (86.6% vs. 78.4%), but the blastocyst developmental rate was significantly increased in the RINT group (43.3% vs. 25.3%; p < 0.01). Blastocysts derived from RINT had a higher total cell number than those from SNT (45.1 +/- 3 vs. 37.6 +/- 4; p < 0.05). Our results provide evidence that RINT is feasible and may provide a more efficient and simple method for NT than INT.

Nucleologenesis and embryonic genome activation are defective in interspecies cloned embryos between bovine ooplasm and rhesus monkey somatic cells

BackgroundInterspecies somatic cell nuclear transfer (iSCNT) has been proposed as a tool to address basic developmental questions and to improve the feasibility of cell therapy. However, the low efficiency of iSCNT embryonic development is a crucial problem when compared to in vitro fertilization (IVF) and intraspecies SCNT. Thus, we examined the effect of donor cell species on the early development of SCNT embryos after reconstruction with bovine ooplasm.ResultsNo apparent difference in cleavage rate was found among IVF, monkey-bovine (MB)-iSCNT, and bovine-bovine (BB)-SCNT embryos. However, MB-iSCNT embryos failed to develop beyond the 8- or 16-cell stages and lacked expression of the genes involved in embryonic genome activation (EGA) at the 8-cell stage. From ultrastructural observations made during the peri-EGA period using transmission electron microscopy (TEM), we found that the nucleoli of MB-iSCNT embryos were morphologically abnormal or arrested at the primary stage of nucleologenesis. Consistent with the TEM analysis, nucleolar component proteins, such as upstream binding transcription factor, fibrillarin, nucleolin, and nucleophosmin, showed decreased expression and were structurally disorganized in MB-iSCNT embryos compared to IVF and BB-SCNT embryos, as revealed by real-time PCR and immunofluorescence confocal laser scanning microscopy, respectively.ConclusionThe down-regulation of housekeeping and imprinting genes, abnormal nucleolar morphology, and aberrant patterns of nucleolar proteins during EGA resulted in developmental failure in MB-iSCNT embryos. These results provide insight into the unresolved problems of early embryonic development in iSCNT embryos.

Effects of IGF-I and EGF Supplemented to PZM3 Culture Medium on the Development of Porcine Embryos In vitro

This study investigated the effects of IGF-I and EGF on the development of blastocysts or hatched blastocysts during the in vitro culture of embryos from immature porcine oocytes. After the in vitro maturation and fertilization of cumulus-oocyte complexes (COCs) and their culture in vitro in PZM3 medium, we examined the embryo development rate for 168 h. When different concentrations of IGF-I (0, 1, 10, 20 ng/ml) were supplemented to fertilized porcine embryos in vitro, there were no significant differences in cleavage rate, blastocyst development rate or blastocyst hatching rate among the treated groups. On the other hand, when different concentrations of EGF (0, 1, 10, 20 ng/ml) were supplemented to the in vitro culture medium, blastocyst development rate was highest in the group in which EGF was not supplemented and, specifically, it was higher than in the 20 ng/ml treatment group (p<0.05). When 10 ng/ml IGF-I and 1 ng/ml EGF were supplemented separately or simultaneously, there were no significant differences among the treated groups in blastocyst hatching rate and the number of cells in each condition. This study demonstrated that the addition of IGF-I and EGF into PZM3 medium did not enhance development of the blastocyst stage and total cell number in blastocysts. (Key Words : Embryo Development, Porcine Embryo, Growth Factor, PZM3)

113 EFFECT OF PLANT PROTEIN ON DEVELOPMENT AND QUALITY OF CULTURED IN VITRO PORCINE ZYGOTES

Basic culture media are usually supplemented with serum albumin or serum, which contain amino acids that play an important role as energy sources, osmoregulators, and pH stabilizers. However, the presence of undefined serum in culture media introduces a variation from batch to batch and increases viral or prion contamination risk. The aim of the present study was to investigate the possibility of using plant protein substitute (PP) in place of bovine serum albumin (BSA) during in vitro culture of porcine zygotes. The PP is a mixture of several plant proteins and soya lecithin prepared using a high pressure homogenization process. The experiment was done on pig zygotes obtained surgically from superovulated gilts at 24–26 h after insemination. Morphologically normal zygotes were cultured in vitro in 5% CO2 in air at 39° in NCSU-23 medium supplemented with: 0.002 g mL–1 (group 1), 0.004 g mL–1 (group 2), 0.008 g mL–1 (group 3) PP or 0.004 g mL–1 BSA (control group). Embryo quality criteria were developmental competence (cleavage, morula and blastocyst rates), total cell number per blastocyst and degree of apoptosis as assessed by TUNEL method. Results were analyzed by Chi-square test. There were no differences in cleavage rates on Day 2 between zygotes cultured in NCSU-23 medium supplemented with PP (86.0, 88.0, 84.8; group 1 to 3, respectively) and BSA (91.0%, control group). Culture with 0.008 g mL–1 PP increased morula (85.7%) and blastocyst (69.2%) production as compared with control (75.0% and 56.3%, respectively; P &lt; 0.05) and 0.002 g mL–1 PP (79.5% and 51.8%, respectively; P &lt; 0.05). The mean number of cells in Day 7 blastocysts cultured in NCSU-23 medium + 0.004 g mL–1 BSA was lower (P &lt; 0.05) than in NCSU-23 + 0.004 g mL–1 PP (39.1 v. 43.7, respectively). The blastocysts cultured in NCSU-23 medium + 0.002 g mL–1 PP had higher average number of apoptotic nuclei (13.0) as compared with the control (6.5) and 0.004 g mL–1 PP (6.9). In conclusion, this study suggest the positive effect of PP on development in vitro of porcine zygotes to the morula/blastocyst stage. However, further studies are required to determine the quality of the embryos cultured with PP. This study was supported by Scientific Net of Animal Reproduction Biotechnology.

109 EFFECTS OF LIPOLYTIC AGENTS FORSKOLIN, EPINEPHRINE AND CAFFEINE ON EMBRYONIC DEVELOPMENT AND LIPID CONTENT OF BOVINE EMBRYOS PRODUCED IN VITRO

The objective of this experiment was to evaluate lipid accumulation and embryonic development of bovine morulae treated with different chemicals. A total of 2619 slaughterhouse oocytes from heifers and mature cows were matured in CDM medium (similar to SOF) plus 0.5% fatty acid-free BSA and hormones (M-CDM) for 23 h at 38.5°C in 5% CO2 in air. Frozen–thawed sperm were centrifuged through a Percoll gradient and co-cultured with matured oocytes for 18 h in F-CDM (CDM+heparin). Zygotes were cultured at 38.5°C in 5% CO2/5% O2/90% N2 in CDM-1 with nonessential amino acids, 10 μm EDTA, 0.5% fatty acid free BSA, and 0.5 mm fructose. After 60 h, resulting 8-cell embryos were cultured 120 h in CDM-2 (CDM-1+essential amino acids and 2 mm fructose). A factorial design was used with 7 treatments, 2 ovary sources (cows v. heifers), and 3 bulls (A, B and C) replicated twice for each bull (6 replicates). At Day 2.5 embryo cleavage and 8-cell rates were evaluated, and on Day 6 a total of 755 morulae were randomly assigned to the 7 treatments (control, 2 and 8 mm caffeine, 1 and 4 μm epinephrine, and 10 and 40 μm forskolin). To quantify lipid accumulation, Day 7 blastocysts were fixed and stained with 1 μg mL–1 Nile red dye, after which a digital photograph of the equatorial part of the embryo (including the inner cell mass) was taken at 200×, and fluorescence intensity was measured with Image Pro software from 0 to 255 shades for each pixel (0 = no lipids; 255 = greatest lipid accumulation), as previously reported (Biol. Reprod. 2007 (Suppl. 1), 87–88). Data were analyzed by ANOVA. No differences in cleavage rates (75 v. 68 ± 3.6%) or eight cell rates (61 ± v. 57 ± 2.8%) were found for heifer v. cow oocytes (P &gt; 0.1); however, blastocyst rates per oocyte and per 8-cell embryo were greater for cows than heifers (20 v. 10 ± 2.1%, and 68 v. 35 ± 3.8%, respectively; P &lt; 0.05). Treatments: 2 and 8 mm caffeine produced fewer blastocysts per morula than 1 and 4 μm epinephrine, 10 and 40 μm forskolin and the control (39, 5 v. 54, 49, 48, 54 and 52 ± 5.8%; respectively) (P &lt; 0.01). More lipid content was found in whole embryos and trophoblast of heifer-derived than cow blastocysts (P &lt; 0.05), and forskolin resulted in less lipid content than control, caffeine- and epinephrine-treated morulae in whole embryos, embryonic mass and trophoblasts (P &lt; 0.05; Table 1). In conclusion, mature cows were a better source of oocytes than feedlot heifers for embryonic development. High doses of caffeine were detrimental to embryos, and the addition of the lypolitic agent forskolin reduced lipid content relative to control, caffeine and epinephrine-treated embryos. Table 1.Main effect treatment means of lipid content (arbitrary fluorescence units)

Developmental competence of immature and failed/abnormally fertilized human oocytes in nuclear transfer

Somatic cell nuclear transfer holds great promise for basic studies of reprogramming human somatic cells and for the potential development of novel cell-based therapeutics. The aim of this study was to examine experimental aspects of human nuclear transfer via use of an abundant source of oocytes, those that are routinely discarded from assisted reproduction clinics. The results suggest and reinforce several findings based on the analysis of multiple parameters: first, it was observed that supplementation of commercial culture media with hormones promoted embryo development after parthenogenetic activation. Second, the use of the chemical activation reagent puromycin resulted in significant differences in cleavage rates in oocytes that were failed/abnormally fertilized after intracytoplasmic sperm injection relative to those from IVF ( P < 0.05). Third, cycloheximide promoted cleavage rates ≥40% in both groups of oocytes; moreover, two blastocysts were produced following cycloheximide treatment. Finally, the use of a subset of oocytes for nuclear transfer resulted in cleaved embryos that expressed green fluorescent protein from a transgene in donor nuclei from human embryonic stem cells. In light of these results, it is suggested that the discarded oocytes can be used to investigate new human nuclear transfer protocols for embryonic stem cell derivation.

Microinjection of mouse phospholipase Cζ complementary RNA into mare oocytes induces long-lasting intracellular calcium oscillations and embryonic development

Methods presently used to activate mare oocytes for assisted reproduction technologies provide low rates of advanced embryonic development. Because phospholipase Czeta (PLCzeta) is the postulated sperm-borne factor responsible for oocyte activation at fertilisation, the aim of the present study was to investigate the pattern of [Ca(2+)](i) oscillations and developmental rates achieved by microinjection of three concentrations of mouse PLCzeta complementary (c) RNA (1, 0.5 or 0.25 microg microL(-1)) into mare oocytes. The frequency of [Ca(2+)](i) oscillations was no different (P > 0.05) after injection of 1, 0.5 or 0.25 microg microL(-1) PLCzeta cRNA (41.1 +/- 5.3, 47 +/- 4.0 and 55.4 +/- 9.0, respectively). However, [Ca(2+)](i) oscillations persisted longest (P < 0.05) for oocytes injected with 0.5 microg microL(-1) PLCzeta cRNA (570.7 +/- 64.2 min). There was no significant difference in cleavage rates after injection of the three concentrations of PLCzeta (P > 0.05; range 97-100%), but the proportion of oocytes reaching advanced stages of embryonic development (>64 nuclei) was significantly lower for oocytes injected with 0.25 microg microL(-1) PLCzeta cRNA (3%) than for those injected with 1 microg microL(-1) PLCzeta cRNA (15%). Based on these results, microinjection of PLCzeta may prove an effective and consistent method for the parthenogenetic activation of mare oocytes for nuclear transfer and provides a physiologically relevant tool with which to study fertilisation-dependent [Ca(2+)](i) signalling in this species.