Effect of MEM vitamins and forskolin on embryo development and vitrification tolerance of in vitro-produced pig embryos

The aims of this study were (1) to determine the effect of in vitro maturation (IVM) medium supplementation with MEM vitamins on in vitro embryo development and sensitivity to vitrification of Day 6 blastocysts and (2) to evaluate whether the addition of forskolin to in vitro culture (IVC) medium enhances blastocyst survival following Super Open Pulled Straw (SOPS) vitrification. Cumulus–oocyte complexes (COCs; n = 4000) were matured with 0.0% or 0.05% (v/v) MEM vitamins. After 44 h of IVM, the oocytes were in vitro fertilized, and presumptive zygotes were cultured. At Day 5 of IVC, embryos from both experimental groups were cultured for 24 h with 0 or 10 μM forskolin, achieving a 2 × 2 factorial design. The blastocyst formation rate was assessed on Day 6, and subsets of samples from the four experimental groups were vitrified (n = 469) or kept fresh (n = 546). Fresh and vitrified-warmed blastocysts were cultured for 24 h prior to embryo survival and total blastocyst cell number assessment. The MEM vitamins increased (P < 0.001) the blastocyst formation rate at Day 6, but they did not affect embryo survival after vitrification. In contrast, the addition of forskolin to the culture medium enhanced (P < 0.05) the blastocyst vitrification tolerance. The total blastocyst cell number was similar among the groups. In conclusion, supplementation with 0.05% MEM vitamins improved the blastocyst formation rate, and the addition of 10 μM forskolin to the culture medium increased survival in Day 6 in vitro-produced blastocysts after SOPS vitrification.     

Ultrasonographic-guided retrieval of cumulus oocyte complexes after super-stimulation in dromedary camel (Camelus dromedarius)

In Experiment 1, studies were conducted to apply the transvaginal ultrasound guided ovum pick-up (OPU) technique in dromedary camels after their ovarian super-stimulation and in vivo oocyte maturation. In Experiment 2, the developmental potential of two commonly used oocyte types, i.e., in vivo matured oocytes collected by OPU and abattoir derived in vitro-matured oocytes was compared after their chemical activation. In Experiment 3, developmental competence of oocytes collected from super-stimulated camels by OPU, matured either in vivo or in vitro, was compared after their chemical activation. Mature female dromedary camels super-stimulated with a combination of eCG and pFSH were given an injection of 20 μg of the GnRH analogue, buserelin 24, 26, or 28 h before the scheduled OPU. For collection of cumulus oocyte complexes (COCs) the transducer was guided through the vulva into the cranial most portion of the vagina and 17-gauge, 55 cm single-lumen needle was placed in the needle guide of the ultrasound probe and advanced through the vaginal fornix and into the follicle. Follicular fluid was aspirated using a regulated vacuum pump into tubes containing embryo-flushing media. Aspirates were searched for COCs using a stereomicroscope, and they were then denuded of cumulus cells by hyaluronidase and repeated pipetting. The oocytes were classified as mature (with a visible polar body), immature (with no visible polar body), activated (with divided or fragmented ooplasm) and others (degenerated and abnormal).Overall an average of 12.12 ± 7.9 COCs were aspirated per animal with an oocyte recovery rate from the aspirated follicles of about 77%. The majority (> 90%) of the collected COCs by OPU were with loose and expanded cumulus cells. The proportion of matured oocytes obtained at 28-29 h (91.2 ± 4.1) and 26-27 h (82.1 ± 3.4) were higher (P < 0.005) when compared with those obtained at 24-25 h (40.4 ± 16.3) after GnRH administration. In Experiment 2, a higher proportion (P < 0.05) of in vivo matured oocytes cleaved (84.6 ± 2.1 vs. 60.9 ± 6.6) and developed to blastocyst stages (52.4 ± 4.1 vs. 30.5 ± 3.3) when compared with in vitro matured oocytes collected from slaughterhouse ovaries. In Experiment 3, no difference was observed between the developmental competences of oocytes, collected from super stimulated camels, matured in vitro with those collected after their in vivo maturation.In conclusion, about 80-90% mature oocytes can be collected by ultrasound guided transvaginal ovum pick-up from super-stimulated dromedary camels 26-28 h after GnRH administration. The developmental response, to chemical activation, of in vivo matured oocytes collected by ultrasound guided transvaginal OPU is better than in vitro matured oocytes obtained from slaughterhouse ovaries. However, no difference was observed in the developmental competence of oocytes collected by OPU whether they were matured in vivo or in vitro.     

The effects of resveratrol on porcine oocyte in vitro maturation and subsequent embryonic development after parthenogenetic activation and in vitro fertilization

We investigated the effects of resveratrol, a phytoalexin with various pharmacologic activities, on in vitro maturation (IVM) of porcine oocytes. We investigated intracellular glutathione (GSH) and reactive oxygen species (ROS) levels, as well as gene expression in mature oocytes, cumulus cells, and in vitro fertilization (IVF)-derived blastocysts, and subsequent embryonic development after parthenogenetic activation (PA) and IVF. After 44 h of IVM, no significant difference was observed in maturation of the 0.1, 0.5, and 2.0 μM resveratrol groups (83.0%, 84.1%, and 88.3%, respectively) compared with the control (84.1%), but the 10.0 μM resveratrol group showed significantly decreased nuclear maturation (75.0%) (P < 0.05). The 0.5- and 2.0-μm groups showed a significant (P < 0.05) increase in intracellular GSH levels compared with the control and 10.0 μM group. Intracellular ROS levels in oocytes matured with 2.0 μM resveratrol decreased significantly (P < 0.05) compared with those in the other groups. Oocytes treated with 2.0 μM resveratrol during IVM had significantly higher blastocyst formation rates and total cell numbers after PA (62.1% and 49.1 vs. 48.8%, and 41.4, respectively) and IVF (20.5% and 54.0 vs. 11.0% and 43.4, respectively) than the control group. Cumulus-oocytes complex treated with 2.0 μM resveratrol showed lower expression of apoptosis-related genes compared with mature oocytes and cumulus cells. Cumulus cells treated with 2.0 μM resveratrol showed higher (P < 0.05) expression of proliferating cell nuclear antigen than the control group. IVF-derived blastocysts derived from 2.0 μM resveratrol-treated oocytes also had less (P < 0.05) Bak expression than control IVF-derived blastocysts. In conclusion, 2.0 μM resveratrol supplementation during IVM improved the developmental potential of PA and IVF porcine embryos by increasing the intracellular GSH level, decreasing ROS level, and regulating gene expression during oocyte maturation.     

Growth and development of rabbit oocytes in vitro: Effect of fetal bovine serum concentration on culture medium

The objective was to develop a culture system that produced blastocyst stage embryos from rabbit oocytes grown in vitro. Two experiments were performed. First, various concentrations of fetal bovine serum (FBS, 0, 0.05, 0.5 and 5%) were used in the culture medium for in vitro growth (IVG) of oocytes recovered from follicles 200 to 299 μm in diameter. Intracytoplasmic sperm injection (ICSI) was performed on mature oocytes obtained after IVG for 8 days and in vitro maturation for 14 to 16 h. Rates of survival and pronuclear formation after ICSI were higher for oocytes grown in a medium with 0.05% FBS compared to oocytes grown in a medium lacking FBS (97.6 vs. 76.9%, 97.5 vs. 70%, P < 0.1). The rate of development to the blastocyst stage was also higher in the medium containing 0.05% FBS than in the medium lacking FBS (9.5 vs. 17.9%, P < 0.05). Next, using oocytes recovered from follicles 200 to 399 μm in diameter which were cultured in 0.05% FBS, oxygen consumption and the number of cells were analyzed. Blastocysts from oocytes grown in vitro with 0.05% FBS had reduced oxygen consumption and number of cells compared with those from ovulated oocytes (21.66 ± 4.54 × 10¹⁴ vs. 50.19 ± 4.61 × 10¹⁴ mol/sec, 244 ± 25 vs. 398 ± 24, P < 0.05). Rabbit oocytes grown in vitro with 0.05% FBS achieved pregnancy, but pregnancies were not maintained to term. In conclusion, the addition of 0.05% FBS to the culture medium for IVG improved developmental competence of rabbit oocytes grown in vitro.     

Improvement in in?vitro fertilization outcome following in?vivo synchronization of oocyte maturation in mice

Synchronization of oocyte maturation in vitro has been shown to produce higher in vitro fertilization (IVF) rates than those observed in oocytes matured in vitro without synchronization. However, the increased IVF rates never exceeded those observed in oocytes matured in vivo without synchronization. This study was therefore designed to define the effect of in vivo synchronization of oocyte maturation on IVF rates. Mice were superovulated and orally treated with 7.5 mg cilostazol (CLZ), a phosphodiesterase 3A (PDE3A) inhibitor, to induce ovulation of immature oocytes at different stages depending on frequency and time of administration of CLZ. Mice treated with CLZ ovulated germinal vesicle (GV) or metaphase I (MI) oocytes that underwent maturation in vitro or in vivo (i.e. in the oviduct) followed by IVF. Superovulated control mice ovulated mature oocytes that underwent IVF directly upon collection. Ovulated MI oocytes matured in vitro or in vivo had similar maturation rates but significantly higher IVF rates, 2–4 cell embryos, than those observed in control oocytes. Ovulated GV oocytes matured in vitro showed similar maturation rates but significantly higher IVF rates than those observed in control oocytes. However, ovulated GV oocytes matured in vivo had significantly lower IVF rates than those noted in control oocytes. It is concluded that CLZ is able to synchronize oocyte maturation and improve IVF rates in superovulated mice. CLZ may be capable of showing similar effects in humans, especially since temporal arrest of human oocyte maturation with other PDE3A inhibitors in vitro was found to improve oocyte competence level. The capability of a clinically approved PDE3A inhibitor to improve oocyte fertilization rates in mice at doses extrapolated from human therapeutic doses suggests the potential scenario of the inclusion of CLZ in superovulation programs. This may improve IVF outcomes in infertile patients.     

Mitochondrial distribution and meiotic progression in canine oocytes during in vivo and in vitro maturation

The objective was to evaluate mitochondrial distribution, and its relationship to meiotic development, in canine oocytes during in vitro maturation (IVM) at 48, 72, and 96 h, compared to those that were non-matured or in vivo matured (ovulated). The distribution of active mitochondria during canine oocyte maturation (both in vitro and in vivo) was assessed with fluorescence and confocal microscopy using MitoTracker Red (MT-Red), whereas chromatin configuration was concurrently evaluated with fluorescence microscopy and DAPI staining. During IVM, oocytes exhibited changes in mitochondrial organization, ranging from a fine uniform distribution (pattern A), to increasing clustering spread throughout the cytoplasm (pattern B), and to a more perinuclear and cortical distribution (pattern C). Pattern A was mainly observed in germinal vesicle (GV) oocytes (96.4%), primarily in the non-matured group (P < 0.05). Pattern B was seen in all ovulated oocytes which were fully in second metaphase (MII), whereas in IVM oocytes, ∼64% were pattern B, irrespective of duration of culture or stage of nuclear development (P > 0.05). Pattern C was detected in a minor percentage (P < 0.05) of oocytes (mainly those in first metaphase, MI) cultured for 72 or 96 h. In vitro matured oocytes had a minor percentage of pattern B (P < 0.05) and smaller mitochondrial clusters in IVM oocytes than ovulated oocytes, reaching only 4, 11, and 17% of MII at 48, 72, and 96 h, respectively. Thus, although IVM canine oocytes rearranged mitochondria, which could be related to nuclear maturation, they did not consistently proceed to MII, perhaps due to incomplete IVM, confirming that oocytes matured in vitro were less likely to be competent than those matured in vivo.     

In vivo and in vitro embryo production in goats

Assisted reproductive technologies (ART) such as artificial insemination (AI) and multiple ovulation and embryo transfer (MOET) have been used to increase reproductive efficiency and accelerate genetic gain. The principal limitations of MOET are due to variable female response to hormonal treatment, fertilization failures and premature regression of Corpora luteum. The in vitro production (IVP) of embryos offers the possibility of overcoming MOET limitations. The method of IVP of embryos involves three main steps: in vitro maturation of oocytes (IVM), in vitro fertilization of oocytes (IVF) with capacitated sperm and in vitro culture (IVC) of embryos up to blastocyst stage. Recovering oocytes from live selected females by laparoscopic ovum pick-up (LOPU) and breeding prepubertal females by juvenile in vitro embryo technology (JIVET) will allow a greater production of valuable goats. Also, IVP of goat embryos will provide an excellent source of embryos for basic research on development biology and for commercial applications of transgenic and cloning technologies. Different protocols of IVP of embryos have been used in goats. However oocyte quality is the main factor for embryos reaching blastocyst stage from IVM/IVF/IVC oocytes. One of the principal determinant factors in the results of blastocyst development is the age of the oocyte donor females. In goats, oocytes from prepubertal and adult females do not show differences in in vitro maturation and in vitro fertilization; however the percentage of oocytes reaching blastocyst stage ranges from 12 to 36% with oocytes from prepubertal and adult goats, respectively.     

The new system of shorter porcine oocyte in vitro maturation (18 hours) using ≥8 mm follicles derived from cumulus-oocyte complexes

Despite recent efforts to improve in vitro maturation (IVM) systems for porcine oocytes, developmental competence of in vitro-matured oocytes is still suboptimal compared with those matured in vivo. In this study, we compared oocytes obtained from large (≥8 mm; LF) and medium (3–7 mm; MF) sized follicles in terms of nuclear maturation, intracellular glutathione and reactive oxygen species levels, gene expression, and embryo developmental competence after IVM. In the control group, cumulus-oocyte complexes (COCs) were aspirated from MF and matured for 22 hours with hormones and subsequently matured for 18 to 20 hours without hormones at 39 °C, 5% CO₂in vitro. In the LF group, COCs were obtained from follicles larger than 8 mm and were subjected to IVM for only 18 hours. The ovaries have LF were averagely obtained with 1.7% per day during 2012 and it was significantly higher in the winter season. The results of the nuclear stage assessment of the COCs from the LFs are as follows: before IVM (0 hours); germinal vesicle stage (15.2%), metaphase I (MI) stage (55.4%), anaphase and telophase I stages (15.8%), and metaphase II (MII) stage (13.6%). After 6 hours IVM; germinal vesicle (4.2%), MI (43.6%), anaphase and telophase I (9.4%), and MII (42.8%). After 18-hour IVM; MI (9.7%) and MII (90.3%). Oocytes from LF showed a significant (P < 0.001) increase in intracellular glutathione (1.41 vs. 1.00) and decrease in reactive oxygen species (0.8 vs. 1.0) levels compared with the control. The cumulus cells derived from LFs showed lower (P < 0.1) mRNA expression of COX-2 and TNFAIP6, and higher (P < 0.1) mRNA expression of PCNA and Nrf2 compared with the control group-derived cumulus cells. After parthenogenetic activation, in vitro fertilization and somatic cell nuclear transfer (SCNT) using matured oocytes from LFs, the embryo development was significantly improved (greater blastocyst formation rates and total cell numbers in blastocysts) compared with the control group. In conclusion, oocytes from LFs require only 18 hours to complete oocyte maturation in vitro and their developmental competence is significantly greater than those obtained from MFs. Although their numbers are limited, oocytes from LFs might offer an alternative source for the efficient production of transgenic pigs using SCNT.     

In vitro developmental competence of prepubertal goat oocytes cultured with recombinant activin-A

The present study was designed to evaluate the effect of activin-A during the in vitro oocyte maturation (IVM) and in vitro embryo culture (IVC) on nuclear maturation, blastocyst yield and blastocyst quality of prepubertal goat oocytes. In Experiment 1, three groups of oocytes were used during the IVM of prepubertal goat oocytes to determine the optimal concentration of recombinant human activin-A added to the maturation medium. Cumulus–oocyte complexes were matured in an IVM medium containing 0, 10 and 100 ng/ml (groups A0, A10 and A100), fertilized and in vitro cultured using standard procedures. In Experiment 2, the addition of 10 ng/ml activin-A at IVM (A10A0), IVC (A0A10) or IVM+IVC (A10A10) was studied and compared with the control group (A0A0). Results of the first experiment demonstrated that the addition of activin-A yielded similar percentages of maturation (⩽71.0%) and blastocyst formation rates (⩽24.9%) than the control group (A0). Experiment 2 showed that exposure of prepubertal goat oocytes to an IVC medium containing 10 ng/ml activin-A (A0A10) significantly increased the rates of development to the blastocyst stage, as compared with the control group (A0A0) (19.5±2.21% v. 13.1±2.37%, respectively; P<0.05). With regard to the blastocyst quality, total number of cells, inner cell mass (ICM) and trophectoderm of prepubertal goat embryos produced in the presence of activin-A did not differ significantly among experimental groups. In summary, these results indicate that supplementation of the IVC medium with activin-A enhances embryo development of prepubertal goat oocytes.     

Improved developmental ability of porcine oocytes grown in nude mice after fusion with cytoplasmic fragments prepared by centrifugation: A model for utilization of primordial oocytes

Primordial oocytes are a potential resource for medical and zoological application, but those of large animals have not yet been reported to show efficient embryonic development. In the present study, we established a pig model for production of blastocysts from primordial oocytes that had been grafted into nude mice and matured in vitro, in combination with fusion of cytoplasmic fragments. Neonatal porcine ovaries in which most follicles are at the primordial stage were minced and grafted into nude mice (Crlj:CD1-Foxn1^nu). About 60 days after detection of vaginal opening, the mice were given 62.5 U/mL porcine FSH for 2 weeks by infusion to enhance follicular development. Developmentally competent oocytes collected from porcine ovaries (conventional oocytes) were matured in vitro and subjected to serial centrifugation to prepare cytoplasmic fragments without a metaphase plate (cytoplasts). Three cytoplasts were fused by electrostimulation to an oocyte retrieved from a host mouse (xenogeneic oocyte) and matured in vitro. Then these fused oocytes were fertilized and subsequently cultured in vitro. No blastocysts were generated from xenogeneic oocytes without fusion of cytoplasm. When xenogeneic oocytes had been fused with three cytoplasts, the blastocyst rate increased significantly to 14.3%, comparable to that for untreated conventional oocytes (20.0%). The numbers of cells in blastocysts for these fused oocytes (37.2 cells/blastocyst) were not significantly different from those for conventional oocytes (25.4 cells/blastocyst). Our findings show that it is possible to use primordial oocytes of large mammals in combination with xenografting of ovarian tissue and also ooplasmic fusion.     

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.     

Successful pregnancy following transfer of feline embryos derived from vitrified immature cat oocytes using 'stepwise' cryoprotectant exposure technique

Oocyte cryopreservation is the desired tool for the ‘long-term’ storage of female genetic potential especially for endangered/valuable species. This study aims at examining the ability of different cryoprotectant (CPA) and CPA exposure techniques to protect immature feline oocytes against cryoinjury during vitrification. Immature oocytes were submitted to different CPA exposure techniques: 1) 2-step DMSO, 2) 4-step DMSO, 3) 2-step EG, 4) 4-step EG, 5) 2-step EG plus DMSO and 6) 4-step EG plus DMSO. Non-CPA treated, non-vitrified oocytes served as controls. The oocytes were then submitted either to in vitro maturation (Experiment 1, n = 334) or to vitrification/warming (Experiment 2, n = 440). The stage of nuclear maturation was subsequently determined. In Experiment 3, the vitrified immature oocytes (n = 254) were matured and fertilized in vitro, and their developmental competence was assessed. A total of 424 embryos derived from vitrified immature oocytes were transferred into the oviduct of 6 recipient queens (Experiment 4).Vitrification reduced significantly the meiotic and developmental competence of immature cat oocytes compared with the non-vitrified controls. The EG alone or a combination of EG and DMSO yielded higher maturation rates than DMSO, irrespective of the CPA equilibration techniques used. The 4-step EG vitrification resulted in the highest maturation rate (37.6%) but cleavage and blastocyst rates were significantly lower than the non-vitrified controls (24.8% and 30.2% vs 62.5% and 49.3%, respectively). Pregnancy was established in recipients receiving embryos derived from non-vitrified and vitrified/warmed immature oocytes. It is concluded that the stepwise CPA exposure technique can be successfully applied for vitrification of immature cat oocytes, in terms of in vitro development but it is likely to affect in utero development.     

端粒酶催化亚基基因在小鼠 胚胎发育早期的转录活性

用 RT-PCR 引物分别扩增成年昆明 (KM) 小鼠睾丸、脾脏、肾脏、肝脏和胸腺组织的总 RNA 发现,端粒酶催化亚基基因 tert 在这些组织中都有转录,目标产物正确组装到 PMD 18-T 载体后测序,结果与已知 cDNA 序列一致 . PMSG/hCG 超数排卵方法获得 KM 小鼠成熟卵母细胞和 CZB 溶液体外培养的胚胎 (KM ♀× KM ♂ ) ,用酸性 Tyrode's 溶液消化透明带后,采用巢式 RT-PCR ,同时分析 tert 基因和持家基因 hprt 的转录发现,对于单个样品来说 , 全部卵母细胞 (15 h-post hCG , 10/10) 都存在 hprt 转录本,其中,只有 40% (4/10) 还同时存在 tert 转录本 . 原核形成初期 (20 h-post hCG , 6/6) 和原核晚期 (30 h post-hCG , 8/8) 的受精卵,以及发育至 2-C 早期的胚胎 (35 h-post hCG , 7/7) 都不转录 tert 基因,只有 hprt mRNA 存在; 2-C 晚期 (50 h-post hCG) 时,两个基因同时转录 (4/8) 和一个基因单独转录 (4/8) 的胚胎各占 50% ;从 4-C 阶段 (65 h-post hCG , 4/4) 开始,包括 8-C 阶段 (75 h-post hCG , 4/4) ,桑椹胚阶段 (93 h-post hCG , 4/4) ,直至囊胚阶段 (118 h-post hCG , 4/4) ,所有的胚胎都同时转录 tert 和 hprt 基因,而且转录水平明显升高 . 以 20 枚胚胎量为模板进行 RT-PCR 发现,原核早期,原核晚期的胚胎中仍然没有 tert 基因转录,只有 hprt mRNA ,但是,在 2-C 早期胚胎中同时检测到了 hprt 和 tert 两种 mRNA. 结果表明,持家基因 hprt 在成熟卵母细胞受精前后,以及胚胎早期发育过程中均存在转录本 . 40% 卵母细胞中存在的 tert mRNA 在受精后很快降解,检测不到;胚胎基因组在 2-C 早期开始转录 tert mRNA ,转录水平逐渐上升 . 结果暗示,小鼠胚胎的基因组 DNA 在 2-C 早期开始启动,功能基因 tert 也在此时开始转录,可能与胚胎发育初期的染色体保护有关 .     

Significant improvement of pig cloning efficiency by treatment with LBH589 after somatic cell nuclear transfer

The low success rate of animal cloning by somatic cell nuclear transfer (SCNT) associates with epigenetic aberrancy, including the abnormal acetylation of histones. Altering the epigenetic status by histone deacetylase inhibitors (HDACi) enhances the developmental potential of SCNT embryos. In the current study, we examined the effects of LBH589 (panobinostat), a novel broad-spectrum HDACi, on the nuclear reprogramming and development of pig SCNT embryos in vitro. In experiment 1, we compared the in vitro developmental competence of nuclear transfer embryos treated with different concentrations of LBH589. Embryos treated with 50 nM LBH589 for 24 hours showed a significant increase in the rate of blastocyst formation compared with the control or embryos treated with 5 or 500 nM LBH589 (32.4% vs. 11.8%, 12.1%, and 10.0%, respectively, P < 0.05). In experiment 2, we examined the in vitro developmental competence of nuclear transfer embryos treated with 50 nM LBH589 for various intervals after activation and 6-dimethylaminopurine. Embryos treated for 24 hours had higher rates of blastocyst formation than the other groups. In experiment 3, when the acetylation of H4K12 was examined in SCNT embryos treated for 6 hours with 50 nM LBH589 by immunohistochemistry, the staining intensities of these proteins in LBH589-treated SCNT embryos were significantly higher than in the control. In experiment 4, LBH589-treated nuclear transfer and control embryos were transferred into surrogate mothers, resulting in three (100%) and two (66.7%) pregnancies, respectively. In conclusion, LBH589 enhances the nuclear reprogramming and developmental potential of SCNT embryos by altering the epigenetic status and expression, and increasing blastocyst quality.     

Supplementation with vascular endothelial growth factor during in vitro maturation of porcine cumulus oocyte complexes and subsequent developmental competence after in vitro fertilization

The aim of the present study was to investigate whether the effects of vascular endothelial growth factor (VEGF) on porcine cumulus oocyte complexes (COCs) and subsequent blastocyst formation following in vitro fertilization are attributable to improved fertilization and cytoplasmic maturation. Porcine COCs were cultured for 42 h in TCM199 medium with 5 ng/mL human recombinant VEGF, and the resultant metaphase II oocytes were fertilized in vitro. COCs without VEGF supplementation served controls. Supplementation with VEGF during in vitro maturation (IVM) significantly (P < 0.05) improved the blastocyst formation rate and total cell number (46.7 ± 3.1% and 82.8 ± 6.7, respectively) compared with controls (32.5 ± 3.4% and 64.1 ± 5.6, respectively). On day 2, the percentage of four-cell stage embryos was significantly higher in the VEGF-matured group (49.1 ± 2.7%) than in the control (33.1 ± 5.8%), and the percentage of two-cell stage embryos was significantly higher in the control group (10.4 ± 1.4%) than in the VEGF-matured group (6.6 ± 0.9%). At 10 h after the onset of in vitro fertilization (IVF), oocytes with two pronuclei were considered as monospermically or normally fertilized, and oocytes with more than two pronuclei were considered as polyspermically fertilized. Monospermy was significantly higher in VEGF-matured oocytes (47.2 ± 4.3%) than in the control (20.0 ± 2.4%), and polyspermy and sperm penetration per oocyte were significantly higher in the control group (54.4 ± 3.8% and 2.3 ± 0.1, respectively) than in the VEGF-matured oocytes (43.9 ± 3.6% and 1.8 ± 0.1, respectively). Supplementation with VEGF during IVM significantly (P < 0.05) improved male pronuclear formation as compared with the control (91.1 ± 1.9 vs 74.4 ± 3.8%). Type III cortical granule distribution in oocytes was more common in VEGF-matured oocytes (78.0%) than in the control (52.1%). These results suggest that VEGF supplementation during IVM enhanced the developmental potential of porcine IVF embryos through higher male pronuclear formation and higher monospermic fertilization rates as a consequence of improved cytoplasmic maturation.     

Comparison of embryo yield and pregnancy rate between in vivo and in vitro methods in the same Nelore (Bos indicus) donor cows

To investigate why the preferred means to produce bovine embryos in Brazil has changed from in vivo to in vitro, we compared these two approaches in the same Nelore cows (n = 30) and assessed total embryo production and pregnancy rates. Without a specific schedule, all cows were subjected to ultrasound-guided ovum pick up (OPU)/in vitro production (IVP) and MOET, with intervals ranging from 15 to 45 d between procedures, respectively. To produce in vivo embryos, cows were superovulated and embryos were recovered nonsurgically from 1 to 3 times (1.4 ± 0.6), whereas OPU/IVP was repeated from 1 to 5 times (3.2 ± 1.2) in each donor cow during a 12-mo interval. Embryos obtained from both methods were transferred to crossbred heifers. On average, 25.6 ± 15.3 immature oocytes were collected per OPU attempt. The average number of embryos produced by OPU/IVP (9.4 ± 5.3) was higher (P < 0.05) than the MOET method (6.7 ± 3.7). However, pregnancy rates were lower (P < 0.05) following transfer of IVP (33.5%) versus in vivo-derived embryos (41.5%) embryos. Embryonic losses between Days 30 and 60 and fetal sex ratio were similar (P > 0.05) between in vivo and in vitro-derived embryos. We concluded that in Nelore cows, with an interval of 15 d between OPU procedures, it was possible to produce more embryos and pregnancies compared to conventional MOET.