Anti-apoptotic effect of melatonin on preimplantation development of porcine parthenogenetic embryos

In the present study, we investigated the effect of melatonin on the preimplantation development of porcine parthenogenetic and somatic cell nuclear transfer (SCNT) embryos. Parthenogenetic embryos were cultured in mNCSU-23 supplemented with various concentrations of melatonin for 7 days. The results revealed that 100 pM was the optimal concentration, which resulted in significantly increased cleavage and blastocyst formation rates. Additionally, 100 pM melatonin provided the highest increase in total cell number of blastocysts. Therefore, the subsequent experiments were performed with 100 pM melatonin. ROS level in 2-8 cell stage embryos in the presence or absence of melatonin was evaluated. Embryos cultured with melatonin showed significantly decreased ROS. Blastocysts cultured with melatonin for 7 days were analyzed by the TUNEL assay. It was observed that melatonin not only increased (P < 0.05) the total cell number but also decreased (P < 0.05) the rate of apoptotic nuclei. Blastocysts cultured with melatonin were assessed for the expression of apoptosis-related genes Bcl-xl and Bax, and of pluripotency marker gene Oct-4 by real-time quantitative PCR. Analysis of data showed that the expression of Bcl-xl was higher (1.7-fold) compared to the control while the expression of Bax was significantly decreased relative to the control (0.7-fold) (P < 0.05). Moreover, the expression of Oct-4 was 1.7-fold higher than the control. These results indicated that melatonin had beneficial effects on the development of porcine parthenogenetic embryos. Based on the findings of parthenogenetic embryos, we investigated the effect of melatonin on the development of porcine SCNT embryos. The results also demonstrated increased cleavage and blastocyst formation rates, and the total cell numbers in blastocysts were significantly higher when the embryos were cultured with melatonin. Therefore, these data suggested that melatonin may have important implications for improving porcine preimplantation SCNT embryo development.     

Embryos aggregation improves development and imprinting gene expression in mouse parthenogenesis

Mouse parthenogenetic embryonic stem cells (PgESCs) could be applied to study imprinting genes and are used in cell therapy. Our previous study found that stem cells established by aggregation of two parthenogenetic embryos at 8‐cell stage (named as a₂PgESCs) had a higher efficiency than that of PgESCs, and the paternal expressed imprinting genes were observably upregulated. Therefore, we propose that increasing the number of parthenogenetic embryos in aggregation may improve the development of parthenogenetic mouse and imprinting gene expression of PgESCs. To verify this hypothesis, we aggregated four embryos together at the 4‐cell stage and cultured to the blastocyst stage (named as 4aPgB). qPCR detection showed that the expression of imprinting genes Igf2, Mest, Snrpn, Igf2r, H19, Gtl2 in 4aPgB were more similar to that of fertilized blastocyst (named as fB) compared to 2aPgB (derived from two 4‐cell stage parthenogenetic embryos aggregation) or PgB (single parthenogenetic blastocyst). Post‐implantation development of 4aPgB extended to 11 days of gestation. The establishment efficiency of GFP‐a₄PgESCs which derived from GFP‐4aPgB is 62.5%. Moreover, expression of imprinting genes Igf2, Mest, Snrpn, notably downregulated and approached the level of that in fertilized embryonic stem cells (fESCs). In addition, we acquired a 13.5‐day fetus totally derived from GFP‐a₄PgESCs with germline contribution by 8‐cell under zona pellucida (ZP) injection. In conclusion, four embryos aggregation improves parthenogenetic development, and compensates imprinting genes expression in PgESCs. It implied that a₄PgESCs could serve as a better scientific model applied in translational medicine and imprinting gene study.     

显微受精废弃的人类卵母细胞体外成熟及孤雌激活

以卵胞浆单精注射(intracytoplasmic sperm injection,ICSI)后废弃的未成熟人类卵母细胞(生发泡期卵母细胞(the germinal vesicle,GV)和第一次减数分裂中期卵母细胞(the metaphase,MI))为材料,使用卵母细胞体外成熟培养液培养未成熟的卵母细胞,分别在人类绒毛膜促性腺激素(human chorionic gonadotrophin,hCG)注射后45、60、84 h观察卵母细胞成熟情况.分别使用钙离子载体(calcium ionophore,CI)A23187联合6-二甲基氨基嘌呤(6-DMAP)法或精子提取物卵胞质内注射(sperm extracts intracytoplasmic injection,SEII)法两种不同的激活方法对体外成熟MII的卵母细胞进行孤雌激活,评价其体外发育潜能.MI卵子体外成熟率要显著高于GV(75.2%vs 30.6%)(P<0.01).与CI/6-DMAP法相比使用SEII/6-DMAP法在激活率(87.5%vs 70.2%)上要明显高于CI/6-DMAP法(P<0.05),但在卵裂率(65.7%vs 72.5%)和桑囊率(0%vs 5.0%)上SEII/6-DMAP法要低于CI/6-DMAP法.注射hCG 45 h组的卵母细胞激活率(91.3%vs 57.9%)、卵裂率(85.7%vs 57.9%)及桑囊率(9.5%vs 0%)均显著高于注射hCG 60 h组(P<0.01).56.8%(117/206)的ICSI废弃的未成熟卵母细胞可以在体外发育成熟,激活后具有一定的发育潜能,卵龄对卵母细胞的质量和发育能力影响较大.     

Responses to experimental fish manipulations in a shallow,hypereutrophic lake: the relative importance of benthic nutrient recycling and trophic cascade

The genetic polymorphism of twelve enzyme systems in two parthenogenetic tetraploidArtemia populations from N. Greece has been studied, using starch gel electrophoresis. The genetic distance within and among the two Greek populations was calculated. The high degree of genetic identity between the populations indicates that they belong to the same species. Each is composed of electrophoretically identifiable clones; the fitness of these clones under different environmental conditions is discussed.     

孤雌生殖累积世代数和雌体年龄对萼花臂尾轮虫繁殖的影响

采用单个体培养方法,研究了孤雌生殖的累积世代数和雌体年龄对萼花臂尾轮虫混交雌体形成和产卵量的影响,结果表明,随着轮虫孤雌生殖累积世代数和增加,各代中的总混交雌体百分率呈减小的趋势,年轮的雌体可产生较多的混交雌体,非混交雌体所产后代中的总混交雌体百分率具有随其祖母年龄的增大而增大的趋势,孤雌生殖的累积世代数对轮对轮虫非混交雌体的平均产卵量无显著的影响,非混交雌体的年龄对其报代的平均产卵量亦无显著的影响。     

Effects of Growth Factors FGF2 and IGF2 on the Development of Parthenogenetic Mouse Embryos in utero and in vitro

We studied the effects of fibroblast growth factor 2 (FGF2) and insulin-like growth factor 2 (IGF2) on the development of parthenogenetic mouse embryos (CBA × C57BK/6)FF₁. The parthenogenetic embryos were treated in vitro during the preimplantation period and, at the blastocyst stage, transplanted into the uterus of pseudopregnant females. The addition of FGF2 at an optimal dose (2.5 ng/ml) to the culture medium increased twofold the number of embryos developed in utero to the somite stages as compared to the control: 18 and 43%, respectively. The parthenogenetic embryos (18–21 somites), treated and nontreated with FGF2 during the preimplantation period, were explanted for further development in vitro and treated with IGF2 at 2.5 g/ml. As a result, many more parthenogenetic embryos (> 87%) of both groups developed in vitro to the stage of 30 or more somites as compared to the control (59%). More than a half of FGF-2-treated parthenogenetic embryos developed to the stage of 40 and some of them, to the stage of 50 somites. The treatment of the parthenogenetic embryos with FGF2 alone at the preimplantation stages did not improve their development in vitro at the postimplantation stages. The results we obtained suggest that the treatment of parthenogenetic embryosin vitro with FGF2 during the preimplantation period increased twofold the number of somite embryos in utero, while their subsequent treatmentin vitro with IGF2 leads to a significant prolongation of their development, as compared to the control.     

Parthenogenesis is self-destructive for scaled reptiles

Parthenogenesis is rare in nature. With 39 described true parthenogens, scaled reptiles (Squamata) are the only vertebrates that evolved this reproductive strategy. Parthenogenesis is ecologically advantageous in the short term, but the young age and rarity of parthenogenetic species indicate it is less advantageous in the long term. This suggests parthenogenesis is self-destructive: it arises often but is lost due to increased extinction rates, high rates of reversal or both. However, this role of parthenogenesis as a self-destructive trait remains unknown. We used a phylogeny of Squamata (5388 species), tree metrics, null simulations and macroevolutionary scenarios of trait diversification to address the factors that best explain the rarity of parthenogenetic species. We show that parthenogenesis can be considered as self-destructive, with high extinction rates mainly responsible for its rarity in nature. Since these parthenogenetic species occur, this trait should be ecologically relevant in the short term.     

RAPD assessment of California phylloxera diversity

Grape phylloxera, Daktulosphaira vitifoliae Fitch, is a parthenogenetic, aphid-like pest which attacks the roots and leaves of many Vitis L. species. Feeding on the roots of V. vinifera L. cultivars leads to decreased plant productivity and eventual death. Isolates of phylloxera have been grouped into ‘biotypes’ based on the aggressiveness of their feeding behaviour, relative reproductive rates, and abilities to cause decline on V. vinifera and the grape rootstock AXR1. The goal of our research was to determine whether these bio-type definitions were reflected in the genetic diversity among laboratory-cultured colonies of Californian phylloxera. Thirteen phylloxera isolates, encompassing biotypes A, B and non-A or -B types based on their feeding and reproduction, were analysed using the RAPD-PCR method. Results indicate that there were genetic differences among isolates and that there was as much polymorphism within biotypes as among biotypes. This is surprising in a parthenogenetic organism and leads us to suggest that there have been multiple introductions of phylloxera into California, that phylloxera are not obligately parthenogenetic, or that there are other mechanisms whereby phylloxera may evolve relatively rapidly.     

The perception of genetic similarity by the solitary parthenogenetic parasitoid Venturia canescens, and its effects on the occurrence of superparasitism

Observations of oviposition patterns adopted by uniparental lines of the solitary parthenogenetic endoparasitoid Venturia canescens Gravenhorst (Hymenoptera: Ichneumonidae) reveal that the occurrence of superparasitism is influenced by the genealogical relationship between adult wasps and conspecific progeny which they encounter within parasitized hosts (larvae of the Indian meal moth Plodia interpunctella Hübner (Lepidoptera: Pyralidae)): the closer the relationship, the lower the occurrence of superparasitism. This behaviour has an adaptive interpretation because it allows Venturia to avoid selective penalties incurred when larval offspring compete with genetically similar progeny. Venturia's ability to discriminate between her own eggs, those of her relatives, and those of other conspecifics is mediated by a chemical marker produced by Dufour's gland, an accessory of the adult female's reproductive system. This conclusion is supported by chemical analyses which reveal that, while Dufour's glands from unrelated females show highly significant variation between the spectra of volatile hydrocarbons contained in their respective secretions, closely-related females show negligible differences in their chemical constitutions. These findings lend further weight to current theory that superparasitism can be deliberately deployed as an adaptive part of a wasp's behavioural repertoire, and also identify the physiological mechanism by which such an oviposition response may be achieved.