A physiological, rather than a superovulated, post-implantation environment can attenuate the compromising effect of assisted reproductive techniques on gene expression in developing mice embryos.

Assisted reproductive techniques (ARTs) may perturb the pre-/peri-conception microenvironments, which subsequently threaten the health of offspring. This study aimed to investigate the effects of superovulation, vitrification, in vitro culture, and embryo transfer on the expression of epigenetic modulators, imprinted genes, and pluripotency markers in expanded blastocysts and Day-9.5 (D9.5) concepti. Results revealed that 53.4% (8/15) and 86.7% (13/15) of genes in the fetus and placenta, respectively, have similar patterns of transcription in all D9.5 concepti, despite the perturbed mRNA expression observed at the blastocyst stage for each embryo-production technique. These observations indicate a counterbalancing of the abnormal expression pattern analyzed at the blastocyst stage during post-implantation development, particularly when the uterus of a naturally synchronized foster mother is employed. Superovulation resulted in the most abnormal expression patterns compared to other treatment groups, although these same blastocysts were able to develop in a synchronized uterus. Thus, superovulation creates a hormonal environment that negatively affected gene expression and impairs fetal growth more adversely during post-implantation development than other ART protocols, such as in vitro culture, vitrification, or embryo transfer-although each did contribute negatively to the implantation and development process. Together, these results may have implications for treating infertility in humans.

Cytoplasmic, rather than nuclear-DNA, insufficiencies as the major cause of poor competence of vitrified oocytes.

To unravel the differential contributions of nuclear-DNA and cytoplasm to the poor 'competence' of oocytes after cryopreservation, reciprocal exchange of metaphase II-spindle chromosomal complex (karyoplast) between vitrified and fresh oocytes was carried out in an ovine animal model. Karyoplast exchange per se was accomplished with high efficiency and in-vitro development of oocytes reconstituted with fresh-karyoplast and vitrified-cytoplast (FK/VC) showed no improvement over VK/VC and control-vitrification oocytes. Blastocyst development of oocytes that were reconstituted with vitrified-karyoplast and fresh-cytoplast (VK/FC) approached that of fresh-controls, however, and was significantly higher than FK/VC, VK/VC, and control-vitrification (all P ≤ 0.05). These results point toward 'cytoplasmic insufficiencies' as the main cause of poor 'competence' of matured oocytes after vitrification.

Effect of epigenetic modification with trichostatin A and S-adenosylhomocysteine on developmental competence and POU5F1-EGFP expression of interspecies cloned embryos in dog.

Adult canine fibroblasts stably transfected with either cytomegalovirus (CMV) or POU5F1 promoter-driven enhanced green fluorescent protein (EGFP) were used to investigate if pre-treatment of these donor cells with two epigenetic drugs [trichostatin A (TSA), or S-adenosylhomocysteine (SAH)] can improve the efficiency of interspecies somatic cell nuclear transfer (iSCNT). Fluorescence-activated cell sorting (FACS), analyses revealed that TSA, but not SAH, treatment of both transgenic and non-transgenic fibroblasts significantly increased acetylation levels compared with untreated relatives. The expression levels of Bcl2 and P53 were significantly affected in TSA-treated cells compared with untreated cells, whereas SAH treatment had no significant effect on cell apoptosis. Irrespective of epigenetic modification, dog/bovine iSCNT embryos had overall similar rates of cleavage and development to 8-16-cell and morula stages in non-transgenic groups. For transgenic reconstructed embryos, however, TSA and SAH could significantly improve development to 8-16-cell and morula stages compared with control. Even though, irrespective of cell transgenesis and epigenetic modification, none of the iSCNT embryos developed to the blastocyst stage. The iSCNT embryos carrying CMV-EGFP expressed EGFP at all developmental stages (2-cell, 4-cell, 8-16-cell, and morula) without mosaicism, while no POU5F1-EGFP signal was observed in any stage of developing iSCNT embryos irrespective of TSA/SAH epigenetic modifications. These results indicated that bovine oocytes partially remodel canine fibroblasts and that TSA and SAH have marginal beneficial effects on this process.

Effects of heat shock during the early stage of oocyte maturation on the meiotic progression, subsequent embryonic development and gene expression in ovine.

Heat shock may affect different aspects of oocyte maturation and its subsequent development to the blastocyst stage. A series of in vitro experiments was performed to determine whether physiologically heat shock (41°C) disrupts the progression of the ovine oocytes through meiosis, activation and blastocyst formation. Cumulus-oocyte complexes (COCs) were aspirated from 2-6-mm follicles and cultured at 38.5°C (control) or 41°C (heat shock) for the first 12 h of maturation. The oocytes were incubated at 38.5°C during the last 10 h of maturation and 8 days after activation. Results showed that most of the oocytes matured under heat-shock conditions remained at the germinal vesicle breakdown (GVBD) stage and they showed an aberrant chromatin configuration. After heat shock, oocyte diameter and time spent for zona pellucida dissolution increased (P < 0.05). The heat-shocked group had a higher percentage of oocytes with incomplete migration of cortical granules (P < 0.05). The heat-shock condition decreased (P < 0.05) cleavage rates (56.19 versus 89.28%) and morula formation (26.85 versus 37.81%). However, there was no significant difference in blastocyst formation and percentage of hatched blastocysts. At 12 h, heat shock had an adverse effect on embryo quality and reduced inner cell mass number (P < 0.05). Quantitative gene expression analysis showed greater transcripts (P < 0.05) for Na/K-ATPase mRNA in heat-shocked oocytes. To sum up, heat shock has disruptive effects on ovine oocyte maturation and can impair cellular and molecular factors that are important for embryo development.

Applicability of universal Bacteroidales genetic marker for microbial monitoring of drinking water sources in comparison to conventional indicators.

Water quality monitoring is essential for the provision of safe drinking water. In this study, we compared a selection of fecal indicators with universal Bacteroidales genetic marker to identify fecal pollution of a variety of drinking water sources. A total of 60 samples were collected from water sources. The microbiological parameters included total coliforms, fecal coliforms, Escherichia coli and fecal streptococci as the fecal indicator bacteria (FIB), Clostridium perfringens and H2S bacteria as alternative indicators, universal Bacteroidales genetic marker as a promising alternative fecal indicator, and Salmonella spp., Shigella spp., and E. coli O157 as pathogenic bacteria. From 60 samples analyzed, Bacteroidales was the most frequently detected indicator followed by total coliforms. However, the Bacteroidales assay failed to detect the marker in nine samples positive for FIB and other alternative indicators. The results of our study showed that the absence of Bacteroidales is not necessarily an evidence of fecal and pathogenic bacteria absence and may be unable to ensure the safety of the water. Further research, however, is required for a better understanding of the use of a Bacteroidales genetic marker as an indicator in water quality monitoring programs.

Nuclear transfer technique affects mRNA abundance, developmental competence and cell fate of the reconstituted sheep oocytes.

The effect of technical steps of somatic cell nuclear transfer (SCNT) on different aspects of cloned embryo development was investigated in sheep. In vitro-matured oocytes were enucleated in the presence or absence of zona and reconstituted by three different SCNT techniques: conventional zona-intact (ZI-NT), standard zona-free (ZF-NT) and intracytoplasmic nuclear injection (ICI-NT). Stepwise alterations in nuclear remodeling events and in mRNA abundances, throughput and efficiency of cloned embryo development and cell allocation of the resulted blastocysts were assessed. Early signs of nuclear remodeling were observed as soon as 2 h post-reconstitution (hpr) for fusion-based methods of nuclear transfer (ZI-NT and ZF-NT) but were not observable until 4 hpr with the ICI-NT method. The relative mRNA abundances of HSP90AA1 (HSP90), NPM2 and ATPase genes were not affected by i) presence or absence of zona, ii) oocyte enucleation method and iii) nuclear transfer method. After reconstitution, however, the relative mRNA contents of POU5F1 (OCT4) with the ZI-NT and ZF-NT methods and of PAPOLA (PAP) with ZF-NT were significantly lower than those for the ICI-NT method. Zona removal doubled the throughput of cloned blastocyst development for the ZF-NT technique compared with ZI-NT and ICI-NT. Cleavage rate was not affected by the SCNT protocol, whereas blastocyst yield rate in ICI-NT technique (17.0±1.0%) was significantly (P<0.05; ANOVA) higher than in ZF-NT (7.1±1.5%) but not in the ZI-NT group (11.2±3.3%). Despite the similarities in total cell number, SCNT protocol changed the distribution of cells in the blastocysts, as ZF-NT-cloned blastocysts had significantly smaller inner cell mass than ZI-NT. These results indicate that technical aspects of cloning may result in the variety of cloning phenotypes.

Kit ligand and glial-derived neurotrophic factor as alternative supplements for activation and development of ovine preantral follicles in vitro.

In vitro growth of preantral follicles has the potential to produce considerable numbers of competent oocytes for use in medicine, agriculture, and even wildlife conservation. The critical regulatory role of growth factors and hormones in the development of preantral follicles has been established. This study investigated the effect of glial-derived neurotropic factor (GDNF) and kit ligand (KL) on the in vitro development of ovine preantral follicles. Results indicated that both GDNF and KL significantly improved activation of primordial follicles, similar to co-addition of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF), which are commonly used for in vitro follicular development. Importantly, GDNF had a more profound effect on follicle health, development, and differentiation compared with KL alone. Furthermore, the combination of GDNF and KL in the presence of EGF and bFGF had a positive, synergic effect on health, development, and differentiation of preantral follicles, as determined by histological and hormonal assessments. The results of this study may provide a foundation for further studies that will unravel the molecular mechanisms of follicular development to further improve the current status of in vitro preantral follicle culture.

Cryosurvival of in vitro produced embryos as affected by health status effect of oocyte donor cow.

In vitro embryo production and embryo vitrification of genetically superior cows that culled inevitably due to health problems can accelerate genetic progress. This study was carried out to investigate whether maternal age and health status effects of high genetic merit cows affect cryosurvival and developmental competence of IVP embryos. In this sense, the effects of ageing and four common culling causes of dairy cows [repeat breeding (RPB), udder problems (UPM), chronic endometritis (CRE), and lameness (LAM)] on in vitro embryo development, and in vivo developmental competence after embryo vitrification were evaluated. The mean number of oocytes obtained per cow did not vary significantly between donors indifferent groups. Cleavage rates in RPB (86.0+/-4.2%), SEN (81.3+/-2.5%) and CRE (77.6+/-6.3%) cows which were comparable to control (95.9+/-1.5%) but were significantly higher than the related rate of UPM donors (50.6+/-2.6%). Importantly, there was no significant difference between the blastocyst rates of different groups. Mean overall survival rate was not different between the groups and was not affected by the blastocyst production rate. There was no significant difference between pregnancy rates of different groups. The results of the present study indicated that in cattle, neither ageing, nor these four diseases affect ovarian potential in terms of the yield and quality of in vitro embryo development.

Specific activation requirements of in vitro-matured sheep oocytes following vitrification-warming.

Oocyte vitrification and assisted oocyte activation have increasingly important roles in assisted reproductive technology. Yet, an important area of concern with matured oocyte cryobiology is that elements of oocytes intimately involved in metaphase-II arrest may be modified by cryopreservation. By comparing different cellular characteristics of unvitrified, vitrified-warmed, and unvitrified-activated oocytes, the present study investigated how vitrification-warming process may affect developmental competence of in vitro-matured sheep oocytes following parthenogenetic activation. Structural, ultrastructural, and molecular analyses indicated that the characteristics of vitrified-warmed oocytes vastly differed from fresh oocytes, instead resembling unvitrified-activated oocytes. For unvitrified oocytes, the highest blastocyst yield (41.8 ± 0.6%) was achieved using the maximum ionomycin concentration (5 µM), and importantly, the duration of ionomycin treatment was not of utmost importance at this concentration. In contrast, the maximum blastocyst yield of vitrified-warmed oocytes (28.4 ± 1.4%) was achieved with a minimal duration of ionomycin treatment (1 min), and further extending the duration dramatically reduced developmental potential of vitrified-warmed oocytes. These results suggested that vitrified-warmed oocytes may need an activation protocol different from unvitrified oocytes. In this respect, unvitrified oocytes were more sensitive to the concentration rather than the duration of ionomycin treatment when compared with vitrified oocytes, which were sensitive to the treatment duration. These results may provide a platform to improve the potential applications of vitrified oocytes in medicine and agriculture.

Vitrification of in vitro produced bovine embryos: effect of embryonic block and developmental kinetics.

In order to investigate whether the kinetics and stage of embryo development affect cryosurvival of in vitro produced bovine embryos, cleaved embryos were categorized in six groups based on their developmental kinetics regarding the stage of embryonic block in bovine (8-16 cell stage): I and II--early (day 2) and late (day 3) 5-8 cell, III and IV--early (day 3) and late (day 4) 8-16 cell, and V and VI--early (day 4) and late (day 5) morula. The cryosurvival and developmental competence of these embryos were compared with each other and also with the corresponding control groups. The potential of 5-8 cell stage embryos to survive vitrification and further develop towards blastocyst stage was significantly lower than vitrified and un-vitrified 8-16 cell and morula stage embryos. These results suggest that, the survival rate and potential of embryos to develop towards blastocyst stage might be affected by the kinetic of the embryo development. Moreover, the results of this study indicated that the optimal stages of early embryo vitrification are post-embryonic block.

Short-term in-vitro culture of goat enriched spermatogonial stem cells using different serum concentrations.

PURPOSE: To investigate the effect of serum supplementing on short-term culture, fate determination and gene expression of goat spermatogonial stem cells (SSCs). METHODS: Crude testicular cells were plated over Datura-Stramonium Agglutinin (DSA) for 1 h, and non-adhering cells were cultured in the presence of different serum concentrations (1, 5, 10, and 15%) for 7 days in a highly enriched medium initially developed in mice. Colonies developed in each group were used for the assessment of morphology, immunocytochemistry, and gene expression. RESULTS: Brief incubation of testicular cells with DSA resulted in a significant increase in the number of cells that expressed the germ cell marker (VASA). The expression of THY1, a specific marker of undifferentiated spermatogonia, was significantly higher in colonies developed in the presence of 1% rather than 5, 10 and 15% serum. CONCLUSION: Goat SSCs could proliferate and maintain in SSC culture media for 1 week at serum concentrations as low as 1%, while higher concentrations had detrimental effects on SSC culture/expansion.

Antioxidant supplementation of culture medium during embryo development and/or after vitrification-warming; which is the most important?

PURPOSE: To determine the most optimal stage for antioxidant supplementation of culture medium to improve developmental competence, cryotolerance and DNA-fragmentation of bovine embryos. METHODS: Presumptive zygotes were first cultured in presence or absence of beta-mercaptoethanol (beta-ME), for 8 days. Subsequently, half of the expanded blastocysts developed in both groups were vitrified, warmed within 30 min and post-warming embryos along with their corresponding non-vitrified embryos were cultured for two further days in presence or absence of (100 microM) betaME. RESULTS: For vitrified and non-vitrified embryos, the best effect was found when betaME was added from day 1 of in vitro culture in continuation with post-warming culture period. Day 1-8 supplementation significantly increased the rates of cleavage, day 7 and day 8 blastocyst production. For non-vitrified embryos, betaME addition during day 1-8 and/or 9-10 of embryo culture improved both hatching rate and quality of hatched embryos. For vitrified embryos, however, the percentage of DNA-fragmentation (18.5%) was significantly higher (p < or = 0.05) than that of embryos developed in absence of betaME but supplemented with betaME during post-warming period (13.5%). CONCLUSIONS: Exogenous antioxidant increases the chance of embryos, even those of fair-quality, to develop to blastocyst. However, antioxidant inclusion during in vitro embryo development is not sufficient to maintain the redox state of these embryos during the critical period of post-warming embryo culture, and therefore, there should be a surplus source of exogenous antioxidant during post-warming embryo culture.

Alpha lipoic acid reverses the negative effect of LPS on mouse spermatozoa and developmental competence of resultant embryos in vitro.

BACKGROUND: Reproductive toxicity of lipopolysaccharide (LPS) on spermatozoa is well established. OBJECTIVE: The aim of the present study was to show the potential benefits of alpha lipoic acid (ALA) as a strong antioxidant in alleviating the reproductive toxicity of LPS. MATERIALS AND METHODS: Sperm cells and cumulus-oocyte complexes (COCs) were collected from healthy NMRI mice (body weights ranged from 25 to 35 g, 100 females and 200 males). Sperm cells were treated with varying doses of ALA (0.01, 0.02, and 0.04 mm) and 0.01 µg/mL of LPS for 4 h. The quality of spermatozoa (ROS production, DNA fragmentation, and spontaneous acrosome reaction), sperm fertilizability, and the consequent developmental competence of oocytes inseminated with ALA/LPS-treated spermatozoa were recorded. RESULTS: The results showed that 0.04 mm of ALA abrogated LPS-reduced sperm motility, viability, ROS production, spontaneous acrosome reaction, fertilizability, and developmental competence. In addition, 0.04 mm ALA significantly reverted the negative effects of LPS on inner cell mass (ICM) cell counts, total cell number (TCM), and ratio between ICM and TCM. DISCUSSION: Our data showed that ALA significantly could abrogate the negative effects of LPS on sperm quality and oocyte developmental competence. Therefore, ALA had the capacity for protecting sperm cells from LPS-induced damage and ensured fertilization and developmental competency. CONCLUSION: These in vitro findings suggested a therapeutic role for ALA in reducing the negative effects of LPS on spermatozoa and early embryonic development.

Optimized combined electrical-chemical parthenogenetic activation for in vitro matured bovine oocytes.

Sperm-mediated oocyte activation is a complex procedure, both in steps and duration, not yet been completely mimicked during in vitro studies, e.g., parthenogenesis or somatic cell nuclear transfer. Furthermore, parthenogenetic studies have been recognized as a suitable model for studying activation efficiency for nuclear transfer cloning. This study, therefore, was conducted to develop an optimized artificial activation method, based on bovine cloning. In vitro matured bovine oocytes were initially exposed to electrical pulse, used for cell fusion during cloning, and then treated with 15 temporal sequential combinations of 3 chemical activators [calcium ionophore (CI), strontium (SR) and ethanol (ET)], followed by exposure to a protein kinase inhibitor or used for in vitro fertilization as control group. Treated and naturally fertilized oocytes were further cultured for up to 8 days. Embryo development was scored daily and blastocyst cell counting was carried out using differential staining at day 8 of culture. Among 15 temporal sequential combinations of three chemical activators, the best cleavage rates were associated with double (SR-CI, 84.4%), triple (CI-SR-ET, 79.4%) and single (CI, 73.7%) compounds, respectively, which were not significantly different with each other and with in vitro fertilized (IVF) (85.5%). The highest blastocyst rates were gained with ET-SR (24.5%), SR-CI-ET (20.4%) and CI (24.5%) accordingly which were not significantly different with each other but significantly lower than IVF (47%). Embryo cell counting further confirmed reasonably better quality of blastocysts produced using double, triple and single compounds. Although most of the sequential artificial activation compounds induced high cleavage rate, close to IVF, but this did not assure comparable further embryo development to the blastocyst stage. Nevertheless, the results suggest exposure of in vitro matured bovine oocytes to electrical pulse, followed by exposure to CI-6-dimethylaminopurine (6-DMAP) or ET-SR-6-DMAP could be regarded as the optimal artificial activation protocol for in vitro development of parthenogenic bovine oocytes or as a step for activation protocol in cloning procedure.

Effects of cilostamide and/or forskolin on the meiotic resumption and development competence of growing ovine oocytes selected by brilliant cresyl blue staining.

The relevance of low developmental competence of in vitro-matured oocyte to the incomplete/delayed cytoplasmic maturation, and the heterogeneity of retrieved oocytes is well established in several species. A short phase of prematuration culture was used to allow better oocyte cytoplasmic maturation. The preselection of growing and fully grown oocytes has been proposed to improve developmental competency. This study investigated the effects of phosphodiesterase type 3-specific inhibitor, cilostamide, and adenylate cyclase activator, forskolin, on the resumption of meiosis and developmental competence of growing ovine oocytes selected by brilliant cresyl blue (BCB) staining. Results indicate that cilostamide, forskolin, and their combination significantly (P < 0.05) increased the percentage of growing (BCB-) oocytes maintained at the germinal vesicle stage. However, only forskolin significantly (P < 0.05) increased the yield and quality of blastocysts derived from BCB- oocytes compared with non-BCB-treated oocytes. We conclude that a short prematuration culture with forskolin may improve the in vitro developmental competency of growing oocytes in ovine.

Developmental competence of ovine oocytes after vitrification: differential effects of vitrification steps, embryo production methods, and parental origin of pronuclei.

Despite many advances in the field of oocyte cryopreservation, the adverse effects of cryopreservation on oocyte competence are still an open challenge in most mammalian species. Using ovine in vitro-matured oocytes, the differential effects of vitrification steps, embryo production methods, and parental origin of pronuclei were systemically investigated to unravel (1) the most critical stage (if any) of oocyte vitrification, (2) the most suitable method (if any) of embryo production for a vitrified oocyte, and (3) differential contributions of male or female pronuclear formation to the poor quality of vitrified oocyte. Although cryoprotectants used during vitrification had some inevitable adverse effects on oocyte competence, the damages caused by low temperature per se (chilling injury) were the main cause of poor quality of vitrified oocytes. When vitrified oocytes underwent either IVF or intracytoplasmic sperm injection (ICSI), embryo development rates were substantially lower than those of fresh ones. In contrast, when vitrified oocytes underwent either parthenogenetic activation (PA) or SCNT, embryo development rates were very similar to those of fresh ones. Evaluation of nuclear morphology after IVF, ICSI, PA, and SCNT oocytes revealed that vitrification had no apparent effect on the female (IVF, ICSI, and PA) and somatic (SCNT) pronuclear formation rates but significantly reduced male pronuclear formation after either IVF or ICSI compared with fresh counterparts. Quantitative analysis of transcripts revealed comparable mRNA abundances of CNX43, HSP90, GMNN, NPM, and OCT4 between vitrified and fresh oocytes, whereas CCNB, ATP1A1, and PAP transcripts were significantly lower in vitrified versus fresh oocytes. Although underlying mechanisms of poor quality of vitrified oocytes are multifactorial, the ability to obtain equivalent development after PA and SCNT, but not IVF and ICSI, in vitrified versus fresh oocytes may argue that the cytoplasm of vitrified oocyte has the necessary components to support in vitro embryonic development of the maternal, even adult somatic cell, chromosomes but fails to do so with sperm chromosomes.

Chemically assisted somatic cell nuclear transfer without micromanipulator in the goat: effects of demecolcine, cytochalasin-B, and MG-132 on the efficiency of a manual method of oocyte enucleation using a pulled Pasteur pipette.

The present study aimed to facilitate widespread application of a previously described manual method of somatic cell nuclear transfer (SCNT) by investigating the effects of demecolcine (a microtubule-depolymerizing chemical), cytochalasin-B (a microfilament-depolymerizing chemical: 2.5µg/ml for 15min) and MG-132 (a proteasome inhibitor chemical) on the (i) incidence of cytoplasmic protrusion of MII chromosomes, (ii) improvement of manual oocyte enucleation, and (iii) in vitro and in vivo developmental competence of SCNT embryos in the goat. Following in vitro maturation, around 65% of goat oocytes contained a characteristic cytoplasmic protrusion of MII-chromosomes. Treatment with demecolcine (0.4µg/ml for 30min) significantly increased this rate to 92.2±4.5%. Treatment with MG-132 (2µM for 30min) could not improve this rate when used alone (61.4±11.5%), but when combined with demecolcine (86.4±8.1%). Treatment with cytochalasin-B completely suppressed this rate whenever used, either alone (7.7±5.1%) or in combination with demecolcine (3.9±1.3%). In a direct comparison, there was no significant difference in quantity and quality of embryos propagated by the manual vs. micromanipulation-based methods of SCNT (cleavage: 85.3±4.5 vs. 89.5±8.9%, blastocyst: 19.5±4.3 vs. 24.3±4.4%, grade 1 and 2 blastocyst: 33.8±7.1 vs. 29.5±6.3%, total cell count: 125±11.1 vs. 122±10.5, respectively). Furthermore, development to live kids at term was not significant between the two SCNT methods. From both technical and economical points of view, the overall in vitro and in vivo efficiency of this manual method of SCNT proved it a simple, fast and efficient alternative for large scale production of cloned goats.

Effect of phosphodiesterase type 3 inhibitor on nuclear maturation and in vitro development of ovine oocytes.

The present study aims to investigate if prematuration culture (PMC) of ovine oocytes in the presence of a phosphodiesterase type 3 (PDE3) inhibitor cilostamide can improve the shortcomings of conventional in vitro maturation (IVM) system. Therefore, a two-step culture system consisting of 22 hours culture in the presence of 1, 10, and 20 µM cilostamide (PMC medium), followed by 22 hours culture in maturation medium, was designed. The effect of cilostamide on gap junction communications and nuclear status was studied. The variables assessed were chromosome organization, spindle pattern, polar body extrusion, and embryonic development. According to the results, inhibition of PDE3 could not permanently block nuclear maturation in ovine oocytes but it delayed the process of nuclear maturation. Elevation of intra-oocyte cAMP concentration could inhibit cumulus cells expansion and maintain gap junction communications between oocyte and cumulus cells. Deletion of cilostamide and refreshing maturation medium after 22 hours culture revealed that cumulus cells were completely expanded. The inhibitory effect induced by 1 µM cilostamide was reversible, and it increased the number of mature oocytes with aligned chromosomes and normal spindle. However, the inhibitory effects of 10 and 20 µM cilostamide was not fully reversible and was associated with deleterious effects on chromosome organization and spindle pattern. Investigation of embryonic development via parthenogenetic activation and in vitro fertilization revealed that the blastocyst rate of oocytes that were prematured with 1 µM cilostamide was not significantly different from oocytes that underwent conventional IVM but it was significantly reduced in oocytes that were prematured with 10 and 20 µM cilostamide. Our results provide the evidence that reduced cAMP via PDE3 is not the only mechanism that controls the progress of nuclear maturation in sheep oocytes, and that alternative or additional mechanisms may also exist.

Cloned sheep blastocysts derived from oocytes enucleated manually using a pulled pasteur pipette.

The potential applications of a simplified method of somatic cell nuclear transfer (SCNT) that is improved in both efficiency and throughput is considerable. Technically, a major step of SCNT is to produce large pools of enucleated oocytes (cytoplasts) efficiently, a process that requires considerable micromanipulation skill and expensive equipment. Here, we have developed an efficient and high-throughput method of manual oocyte enucleation using a simple device, a pulled Pasteur pipette, that can be connected to standard zona-free method of embryo reconstitution. Common Pasteur pipettes were pulled on a flame to produce finely drawn pipettes with inner diameters approximately less than half the oocyte diameter (∼50-60 µm), and slightly larger than cytoplasmic protrusion (∼20-30 µm) that was induced after demecolcine treatment of MII-stage oocytes. Oocyte manipulation was performed under a stereomicroscope by either bisecting the oocyte into two approximately equal demioocytes (blind manual enucleation), or by positioning the oocytes so that the cytoplasmic extrusion that contains the MII chromosome mass is removed with the minimum amount of cytoplasm (oriented manual enucleation). The survival rate of the manually enucleated oocytes was 81.4-91.5%, comparable to standard zona-free method of oocyte enucleation (>95%). A total of 80-120 oocytes could be enucleated in 10 min, which was considerably higher than standard zona-free enucleation method. In vitro development rates of cloned embryos derived from manually enucleated cytoplasts with varying cytoplasmic volumes (50%, 95%, and 100%) was comparable, and embryonic developmental rates of the two latter groups were at least as good as standard zona-free method. The manual method of oocyte enucleation described here can be learned and mastered for simple, fast, and cheap production of cloned embryos with comparable efficiency to other available methods.

Specific activation requirements of zona-free sheep oocytes before and after somatic cell nuclear transfer.

In this study, the effect of the steps involved in zona-free somatic cell nuclear transfer (SCNT) on oocyte transcripts was investigated in sheep. To establish the reliable combined electrical-chemical activation for zona-free oocytes, oocytes were first exposed to an electrical pulse and then treated with 18 chemical activation regimens designed through modifying duration and concentration of ionomycin and 6-dimethyl aminopurine (6-DMAP), which is routinely used for SCNT. Electrofusion-mediated nuclear transfer significantly reduced transcript abundances of CCNB1, POU5F1, NPM2, GMMN, and CX43 compared to intact oocytes. Maximum parthenogenetic blastocyst development was obtained when oocytes were submitted to electric pulse and then to (1) 5 µM ionomycin for 5 or 2.5 min, both followed by 2 h of incubation with 6-DMAP (41.7±1.1, and 42.4±1.4%, respectively), (2) 5 µM ionomycin for 1 min+6-DMAP for 4 h (43.1±1.4%), and (3) 2.5 µM ionomycin for 1 min+6-DMAP for 2 h (42.4±1.4%), with significant differences compared to all the other groups. Statistical assessment of interactions between duration and concentration of ionomycin and duration of 6-DMAP exposure revealed that (1) concentration of ionomycin may be a more important factor than its duration, (2) both a long exposure period and a low concentration of ionomycin had marked decreasing effects on parthenogenetic development of zona-free oocytes, and (3) high duration of exposure to 6-DMAP can reduce parthenogenetic development. Despite an activation preference of parthenogenetic oocytes, a significantly higher rate of cloned blastocyst development was observed when reconstructed oocytes were activated with 5 µM ionomycin for 5 min rather than 2.5 µM ionomycin for 1 min (8.8±2.5 vs. 1.25±2.2%). These results suggested that SCNT steps have determining effects on oocyte transcripts and activation preferences of the reconstituted oocytes compared to intact counterparts. In this sense, reconstituted oocytes may need a higher concentration of ionomycin for a longer period than intact oocytes.