Effects of exogenous double-stranded RNA on the basonuclin gene expression in mouse oocytes

In plants and less-advanced animal species, such as C.elegans, introduction of exogenous double-stranded RNA (dsRNA) into cells would trigger degradation of the mRNA with homologous sequence and interfere with the endogenous gene expression. It might represent an ancient anti-virus response which could prevent the mutation in the genome that was caused by virus infection or mobile DNA elements insertion. This phenomenon was named RNA interference, or RNAi. In this study, RNAi was used to investigate the function of basonuclin gene during oogenesis. Microinjection of dsRNA directed towards basonuclin into mouse germinal-vesicle-intact (GV) oocytes brought down the abundance of the cognate mRNA effectively in a time- and concentration-dependent manner. This reduction effect was sequence-specific and showed no negative effect on other non-homologous gene expression in oocytes, which indicated that dsRNA can recognize and cause the degradation of the transcriptional products of endogenous basonuclin gene in a sequence-specific manner. Immunofluorescence results showed that RNAi could reduce the concentration of basonuclin protein to some extent, but the effect was less efficient than the dsRNA targeting towards tPA and cMos which was also expressed in oocytes. This result might be due to the long half life of basonuclin protein in oocytes and the short reaction time which was posed by the limited life span of GV oocytes cultured in vitro. In summary, dsRNA could inhibit the expression of the cognate gene in oocytes at both mRNA and protein levels. The effect was similar to Knock-out technique which was based on homologous recombination. Furthermore, hairpin-style dsRNA targeting basonuclin gene could be produced by transcription from a recombinant plasmid and worked efficiently to deplete the cognate mRNA in oocytes. This finding offered a new way to study the function of basonuclin in the early stage of oogenesis by infection of primordial oocytes with the plasmid expressing hairpin-style basonuclin dsRNA.

Vitrification of Yunnan Yellow Cattle oocytes: work in progress

The objective of this study was to provide a simple cryopreservation method for oocytes from Yunnan Yellow Cattle and facilitate preservation efforts in this native Chinese breed, which is threatened by agricultural modernization. Cumulus-oocyte complexes (COCs) were collected from slaughterhouse ovaries and matured in vitro for 22-24 h, then selected for cryopreservation. Vitrification in open pulled straws (OPS) or in microdrops on a cooled metal surface (solid surface vitrification, SSV) was compared. The OPS vitrification solution consisted of 20% ethylene glycol (EG) and 20% DMSO. The SSV solution was a mixture of 35% EG, 5% polyvinyl-pyrrolidon (PVP) and 0.4 M trehalose. Vitrified and warmed oocytes were either fertilized in vitro or parthenogenetically activated. The rates of cleavage and development to blastocysts of fertilized oocytes following OPS versus SSV were not statistically different (38.3 and 12.5% versus 35.8 and 6.0%, respectively). The corresponding rates of parthenogenetic development to blastocysts were also not different (8.2 versus 3.5%, respectively). Development to blastocysts of non-vitrified controls following fertilization was significantly higher than that of the vitrified oocytes (22.6%, P < 0.05). These results demonstrate for the first time, that although both OPS and SSV procedures reduced embryonic development, Yunnan Yellow Cattle oocytes are capable of developing to blastocysts following cryopreservation. (C) 2002 Elsevier Science Inc. All rights reserved.

Amino acid requirements for maturation of rhesus monkey (Macacca mulatta) oocytes in culture

This study evaluated the effects of different amino acid formulations on supporting meiotic and cytoplasmic maturation of rhesus monkey (Macacca mulatta) oocytes in vitro. Five hundred and forty-six cumulus-oocyte complexes (COCs) aspirated from unstimulated adult monkey follicles (greater than or equal to 1000 mum in diameter) were cultured in either modified Connaught Medical Research Laboratories 1066 medium (mCMRL-1066) or in one of eight chemically defined media (modified basic medium 5 supplemented with 5.5 mmol glucose l(-1), 0.003 mmol pantothenic acid l(-1) and different amino acid formulations) as below: (1) modified basic medium 5 (mBM5) containing no amino acid; (2) mBM5 + 0.2 mmol glutamine l(-1); (3) mBM5 + 11 amino acids from hamster embryo culture medium 6 (HECM-6) (11 AA); (4) mBM5 + Eagle's non-essential amino acids (NEA); (5) mBM5 + NEA + 0.2 mmol glutamine l(-1); (6) mBM5 + Eagle's essential amino acids (EA) without glutamine; (7) mBM5 + EA + 0.2 mmol glutamine l(-1); (8) mBM5 + Eagle's 20 amino acids (20 AA) + 0.2 mmol glutamine l(-1); and (9) mCMRL-1066 (control). All media contained FSH, LH, oestradiol and progesterone. After maturation, mature oocytes were subjected to the same fertilization and embryo culture procedures. COCs matured in treatment 5 had greater potential to progress to metaphase II (66%; P < 0.05) than did those in treatments 1 (37.3%), 2 (48.3%)f 3 (41%), 6 (41%) and 9 (43%). Oocytes matured in treatment 8 had the best morula (53%) and blastocyst (18%) developmental responses (P<0.05). The lowest (P<0.05) morula and blastocyst developmental responses were obtained from COCs matured in treatments 1 (0%) and 6 (8%). The other media supported intermediate embryonic development (range 11-38% of morula and blastocyst). These results indicate that the choice of amino acids affects the competence of oocyte maturation and that Eagle's 20 AA with 0.2 mmol glutamine l(-1) is more efficient than the other amino acid formulations for maturation of rhesus monkey oocytes.

Molecular phylogeography and population structure of a mid-elevation montane frog Leptobrachium ailaonicum in a fragmented habitat of southwest China

Leptobrachium ailaonicum is a vulnerable anuran restricted to a patchy distribution associated with small mountain streams surrounded by forested slopes at mid-elevations (approximately 2000-2600 m) in the subtropical Mount Wuliang and Mount Ailao ranges in southwest China (Yunnan Province) and northern Vietnam. Given high habitat specificity and lack of suitable habitat in lower elevations between these ranges, we hypothesized limited gene flow between populations throughout its range. We used two mitochondrial genes to construct a phylogeographic pattern within this species in order to test our hypothesis. We also examined whether this phylogeographic pattern is a response to past geological events and/or climatic oscillations. A total of 1989 base pairs were obtained from 81 individuals of nine populations yielding 51 unique haplotypes. Both Bayesian and maximum parsimony phylogenetic analyses revealed four deeply divergent and reciprocally monophyletic mtDNA lineages that approximately correspond to four geographical regions separated by deep river valleys. These results suggest a long history of allopatric separation by vicariance. The distinct geographic distributions of four major clades and the estimated divergence time suggest spatial and temporal separations that coincide with climatic and paleogeographic changes following the orogeny and uplift of Mount Ailao during the late Miocene to mid Pliocene in southwest China. At the southern distribution, the presence of two sympatric yet differentiated clades in two areas are interpreted as a result of secondary contact between previously allopatric populations during cooler Pleistocene glacial cycles. Analysis of molecular variance indicates that most of the observed genetic variation occurs among the four regions implying long-term interruption of maternal gene flow, suggesting that L ailaonicum may represent more than one distinct species and should at least be separated into four management units corresponding to these four geographic lineages for conservation. (C) 2009 Elsevier Inc. All rights reserved.

SPECIES DIVERSITY DYNAMICS OF FRAGMENTED TROPICAL RAINFORESTS IN THE LOWER-LANCANG/ UPPER-MEKONG RIVER BASIN

　在澜沧江下游/ 湄公河上游的滇南西双版纳地区,通过样方法比较了热带雨林的连片与3 个小片断的物 种多样性变化趋势。与连续森林比较,片断热带雨林的植物物种丰富度和物种多样性指数都比较低,而且有相当低 比例的大高位芽、中高位芽和附生等生活型植物,而藤本、小高位芽和矮高位芽等生活型植物的比例则较高;泛热 带、热带亚洲至热带非洲的区系成分比例较高,而当地成分则减少;群落的上层树木比下层树木更加稳定。同样,动 物的物种多样性指数和均衡度在片断热带雨林中都较低,与其密切相关的是片断热带雨林的环境质量,而不是片 断的大小。此外,也探讨了片断热带雨林物种变化与森林小气候的关系,阐明了由凉湿向干暖转化的“林内效应"是 其物种变化的重要原因之一。

Histone H2A Has a Novel Variant in Fish Oocytes

Histone variants and their modification have significant roles in many cellular processes. In this study, we identified and characterized the histone H2A variant h2af1o in fish and revealed its oocyte-specific expression pattern during oogenesis and embryogenesis. Moreover, posttranslational modification of H2af1o was observed that results from phosphorylation during oocyte maturation. To understand the binding dynamics of the novel core histone variant H2af1o in nucleosomes, we cloned ubiquitous gibel carp h2afx as a conventional histone control and investigated the dynamic exchange difference in chromatin by fluorescence recovery after photobleaching. H2af1o has significantly higher mobility in nucleosomes than ubiquitous H2afx. Compared with ubiquitous H2afx, H2af1o has a tightly binding C-terminal and a weakly binding N-terminal. These data indicate that fish oocytes have a novel H2A variant that destabilizes nucleosomes by protruding its N-terminal tail and stabilizes core particles by contracting its C-terminal tail. Our findings suggest that H2af1o may have intrinsic ability to modify chromatin properties during fish oogenesis, oocyte maturation, and early cleavage.

Microsatellite diversity and population genetic structure of redfin culter (Culter erythropterus) in fragmented lakes of the Yangtze River

Redfin culter (Culter erythropterus) is a small lethic freshwater fish and widely distributed in the adjacent lakes of the Yangtze River of China. Five microsatellite loci were applied to investigate the genetic variation and population structure of redfin culter from seven lakes in the middle-and-lower reaches of the Yangtze River. The gene diversity was high among the populations (H > 0.9), the average number of alleles among seven populations was low with a range from 2.00 to 3.87. The mean observed (H-O) and expected (H-E) heterozygosity ranged from 0.111 to 0.419 and from 0.162 to 0.750, respectively. Significant deviations from Hardy-Weinberg Equilibrium expectation were found in 50% of the total locus-population combination tests in which heterozygote deficits were apparent. The analysis of molecular variance (AMOVA) indicated that the percentage of variance among and within these populations were 6.18 and 93.82, respectively. The Fst values (0.062, P < 0.001) among studied populations indicated that there were significant genetic differentiations among redfin culture populations from the scattered lakes with different connections to the Yangtze River. These results are useful for the evaluation and conservation of small freshwater fishes. The factors that may be involved in low intra-population polymorphism and the pattern of the population genetic structure of redfin culter from the Yangtze River were discussed.

cDNA cloning and characterization of a novel SNX gene differentially expressed in previtellogenic oocytes of gibel carp

To understand the molecular events governing fish oogenesis, a multiple technique was used to identify the genes differentially expressed at different phases during fish oogenesis. This technique is a combination of suppression subtractive hybridization, SMART cDNA synthesis and RACE-PCR. Here we report the cDNA cloning and expression characterization of a novel SNX gene based on its differential transcription between previtellogenic and fully mature oocytes in naturally gynogenetic gibel carp. First, a cDNA fragment selectively expressed in previtellogenic oocytes was identified and used to screen a SMART cDNA library prepared from the same mRNA sample by RACE-PCR for cloning fully length cDNA. The full length cDNA was 1392-bp long and coded for a novel SNX protein with 225 amino acids. The 5' UTR had 72 bp and 3' UTR had 642 bp. Unlike most of maternal genes that are transcribed after vitellogenesis and stored in oocytes, this gene is expressed at a higher level in the previtellogenic oocytes and at a much lower level in fully matured oocytes. However, RT-PCR analysis of tissues showed it was ubiquitous transcription. The novel gene is named fish sorting nexin (fSNX), because it contains a conserved PX domain. The fact which major expression of the gene occurs in the previtellogenic oocytes suggests that it might have an important function in the oogenesis. (C) 2003 Elsevier Inc. All rights reserved.

Differential gene expression in fully-grown oocytes between gynogenetic and gonochoristic crucian carps

Silver crucian carp (Carassius auratus gibelio) is a unique triploid bisexual species that can reproduce by gynogenesis. As all other gynogenetic animals, it keeps its chromosome integrity by inhibiting the first meiosis division (no extrusion of the first pole body). To understand the molecular events governing this reproduction mode, suppression subtractive hybridization was used to identify the genes differentially expressed in fully-grown oocytes of the gynogenetic and gonochoristic crucian carp (gyno-carp and gono-carp). From two specific subtractive cDNA libraries, the clones screened out by dot blots and virtual Northern blots were chosen to clone, full-length cDNA by RACE. Four differentially expressed genes were obtained. Two are novel genes and are expressed specifically in the oocytes. The gyno-carp stores much more mRNA of cyclin A2, a new member of the fish A-type cyclin gene, in its fully-grown oocyte than in the gono-carp. The last gene is histone H2A. The histone H2As of these two closely related crucian carps are quite different in the C-terminus. Preliminary characterization of the four genes has been analyzed by nucleotide and deduced amino acid sequence and Northern analysis. (C) 2001 Elsevier Science B.V. All rights reserved.

Differential gene expression of protein kinases in oocytes between natural gynogenetic silver crucian carp and amphimictic crucian carp

A modified mRNA differential display method has been applied to studying differential expression of protein kinase genes in oocytes between natural gynogenetic silver crucian carp and amphimictic crucian carp. Total RNA was reverse transcribed using downstream 3' primers T(12)MA, T(12)MG and T12MC respectively. Then the reverse transcription products were amplified using upstream 5' kinase-specific primer designed according to protein kinase conserved sequence. The PCR products had different patterns and numbers of: cDNA bands on polyacrylamide:gel. Totally 21 cDNAs fragments were recovered and cloned. Two of them were confirmed to be particularly expressed in oocytes of amphimictic crucian carp, and another was specific for gynogenetic silver crucian carp.

Chromosomal aberrations induced by C-12(6+) ions and Co-60 gamma-rays in mouse immature oocytes

The ovaries of Kun-Ming strain mice (3 weeks) were irradiated with different doses of C-12(6+) ion or Co-60 gamma-ray. Chromosomal aberrations were analyzed in metaphase II oocytes at 7 weeks after irradiation. The relative biological effectiveness (RBE) of C C-12(6+) ion was calculated with respect to Co-60 gamma-ray for the induction of chromosornal aberrations. The C-12(6+) ion and Co-60 gamma-ray dose-response relationships for chromosomal aberrations were plotted by linear quadratic models. The data showed that there was a dose-related increase in frequency of chromosomal aberrations in all the treated groups compared to controls. The RBE values for C-12(6+) ions relative to (CO)-C-60 gamma-rays were 2.49, 2.29, 1.57, 1.42 or 1.32 for the doses of 0.5, 1.0, 2.07 4.0 or 6.0 Gy, respectively. Moreover, a different distribution of the various types of aberrations has been found for C-12(6+) ion and Co-60 gamma-ray irradiations. The dose-response relationships for C-12(6+) ion and (CO)-C-60 gamma-ray exhibited positive correlations. The results from the present study may be helpful for assessing genetic damage following exposure of immature oocytes to ionizing radiation.

Role of animal pole protuberance and microtubules during meiosis in sea cucumber Apostichopus japonicus oocytes

Fully grown oocytes of Apostichopus japonicus have a cytoplasmic protuberance where the oocyte attaches to the follicle. The protuberance and the oolamina located on the opposite side of the oocyte indicate the animal-vegetal axis. Two pre-meiotic centrosomes are anchored to the protuberance by microtubules between centrosomes and protuberance. After meiosis reinitiation induced by DTT solution, the germinal vesicle (GV) migrates towards the protuberance. The GV breaks down after it migrates to the oocyte membrane on the protuberance side. The protuberance then contracts back into the oocyte and the first polar body extrudes from the site of the former protuberance. The second polar body forms beneath the first. Thus the oocyte protuberance indicates the presumptive animal pole well before maturation of the oocyte.

Hardening of zona pellucida of mouse oocytes and embryos in vivo and in vitro

Objective - To evaluate the effect of in vitro culture on zona pellucida resistance in mouse oocytes and embryos. Method-Zona pellucida resistance was assessed by comparing duration of zona lysis in the presence of alpha- chymotrypsin. The effects of artificial or physiological conditions of development were evaluated by comparing embryos in vitro with those left to reach the same stage of development in vivo. Results - The time required for zona lysis of oocytes increased after 2, 9.4, and 48 hours in vitro (P < .001). The same observation holds true for oocytes left in vivo during 24 hours. Fertilization both in vivo and in vitro induced a major increase in zona resistance. At the two-cell stage, in vitro culture did not harden the zona pellucida. At the morula stage and beyond, enzymatic lysis was slightly longer in vitro as compared to that of similar stages recovered from the genital tract. Conclusions - Our data indicate that in vitro culture conditions do not modify zona hardening in oocytes and only slightly increased zona resistance from the morula stage on.

Features of programmed cell death in intact Xenopus oocytes and early embryos revealed by near-infrared fluorescence and real-time monitoring.

Factors influencing apoptosis of vertebrate eggs and early embryos have been studied in cell-free systems and in intact embryos by analyzing individual apoptotic regulators or caspase activation in static samples. A novel method for monitoring caspase activity in living Xenopus oocytes and early embryos is described here. The approach, using microinjection of a near-infrared caspase substrate that emits fluorescence only after its proteolytic cleavage by active effector caspases, has enabled the elucidation of otherwise cryptic aspects of apoptotic regulation. In particular, we show that brief caspase activity (10 min) is sufficient to cause apoptotic death in this system. We illustrate a cytochrome c dose threshold in the oocyte, which is lowered by Smac, a protein that binds thereby neutralizing the inhibitor of apoptosis proteins. We show that meiotic oocytes develop resistance to cytochrome c, and that the eventual death of oocytes arrested in meiosis is caspase-independent. Finally, data acquired through imaging caspase activity in the Xenopus embryo suggest that apoptosis in very early development is not cell-autonomous. These studies both validate this assay as a useful tool for apoptosis research and reveal subtleties in the cell death program during early development. Moreover, this method offers a potentially valuable screening modality for identifying novel apoptotic regulators.