龙眼体细胞胚胎高质量浓度蔗糖成熟培养后的超微结构变化

以龙眼红核子品种的松散型胚性愈伤组织诱导的体细胞胚胎为材料 ,研究了体细胞胚胎经 5 0 g· L- 1 蔗糖成熟培养后的超微结构变化 .结果表明 ,经过成熟培养之后 ,龙眼体细胞胚胎细胞在结构和代谢上都发生了根本性的变化 .该研究有助于了解龙眼体细胞成熟机理以及进一步优化龙眼体胚再生系统     

CK1 inhibitor affects in vitro maturation and developmental competence of bovine oocytes

The objectives of present study were to evaluate the effect of casein kinase 1 (CK1) inhibition D4476 on in vitro maturation (IVM) and developmental competence of bovine oocytes. The cumulus oocyte complexes (COCs) were cultured in maturation medium with D4476 (0, 2, 5, 10, 20 μM) for 24 hr. After IVM and in vitro fertilization, through expansion average scores of cumulus cells (CCs), oocyte maturation efficiency, cleavage rate and blastocyst rate of zygote, we found 5 μM D4476 could increase the development potential of oocytes. After the COCs were treated with 5 μM D4476, the results of quantitative real‐time PCR analysis, Lichen red staining and PI staining showed that under without affecting germinal vesicle breakdown and nuclear morphology, D4476 could significantly decrease CK1 and upregulate TCF‐4 in oocytes. Furthermore, without influencing the level of Bad and CTSB, D4476 could significantly increase the expression of β‐catenin, TCF‐4, Cx43, MAPK, PTGS‐2, PTX‐3, TGS‐6, Bax and Bcl‐2 in CCs. Western blot analysis revealed that the addition of 5 μM D4476 during the maturation of COCs resulted in a lower level of Cx43 protein at 12 hr and a higher expression of Cx43 protein at 24 hr compared to the group without D4476. These results indicate that adding optimum D4476 (5 μM) to maturation medium is beneficial to maturity efficiency and development competence of bovine oocytes.     

Density effects on length at maturity of walleye pollock <Emphasis Type="Italic">Theragra chalcogramma</Emphasis> off the pacific coast of northern Japan in the 1990s

To determine how stock abundance fluctuations of walleye pollock Theragra chalcogramma are related to variations in reproductive characteristics, the length at 50% maturity of the fish sampled off the Pacific coast of northern Japan from 1990 to 1999 was examined. In both sexes, the fish density increased, and the body length of age-3-5 fish decreased, but the condition factor and the age at 50% maturity showed no clear trend during this period. Male length at 50% maturity decreased, while value for females showed no clear trend. In both sexes, significant negative correlations were found between density index (combined age-3-5) and each body length at age-3-5. Significant positive correlations were seen between each body length at age-3-5 and length at 50% maturity in males. Positive correlation between body length at age-5 and length at 50% maturity was significant in females from 1990 to 1999, except for the samples in 1998. We suggest that the fish density might affect the length at maturity in males and possibly also in females.     

谷氨酰胺对延边黄牛卵母细胞成熟及重组胚发育的影响

为了提高卵母细胞的成熟率及重组胚胎的发育率,在体外成熟液中添加了不同浓度的谷氨酰胺,研究谷氨酰胺对延边黄牛卵母细胞成熟的影响,并观察它对重组胚胎后续发育的影响,从中筛选出最佳的体外培养条件。结果表明,1.0 mmol/L的谷氨酰胺能够显著提高卵母细胞的成熟率,同时也能提高囊胚发育率。说明谷氨酰胺能够提高延边黄牛卵母细胞的体外成熟率,并能促进囊胚发育。     

胰岛素和白血病抑制因子对猪卵母细胞体外成熟和孤雌激活胚胎的影响

为探讨胰岛素(Insulin)和白血病抑制因子(Leukemia inhibit factor,LIF)对猪卵母细胞体外成熟(IVM)和猪孤雌激活胚胎(PAEs)的影响,在卵母细胞体外成熟或者胚胎培养基中添加Insulin和LIF,研究卵裂率和囊胚率的变化。结果:添加了5μg/mL Insulin后猪卵母细胞体外成熟效果显著提高,但成熟后孤雌激活发育能力与非添加组相近;而胚胎培养基中添加Insulin对孤雌胚的卵裂和囊胚的形成也没有明显促进作用;添加1 000 U/mL的LIF后,卵母细胞核成熟率没有明显提高,反而孤雌激活后囊胚率急剧下降,但对卵裂率以及囊胚总细胞数影响不大;在胚胎培养基中添加LIF后,孤雌胚的卵裂和囊胚形成并没有明显的提高。表明:Insulin对卵母细胞体外成熟有益,但是对孤雌胚胎的最佳处理程序还需要摸索;本文所采用的LIF处理对猪卵体外成熟以及孤雌胚胎体外发育没有帮助,还需要进一步研究其他浓度和处理程序对猪卵母细胞体外成熟和孤雌激活胚胎发育能力的影响。     

Effects of steroid hormones on in vitro oocyte maturation in white sturgeon (Acipenser transmontanus)

The in vitro effects of several steroids on the maturation of intact white sturgeon (Acipenser transmontanus) ovarian follicles were investigated. At the highest concentration (1024 ng ml^–1 for the C21 steroids and 1139 ng ml^–1 for the C19 steroids), all of the C21 steroids tested, progesterone (P4), 17-hydroxyprogesterone (17OHP), 17,20-dihydroxy-4-pregnen-3-one (17,20-P), 17,(20,21-trihydroxy-4-pregnen-3-one 20-S), 11-deoxycortisol (S) and cortisol (F), as well as testosterone (T) induced germinal vesicle breakdown (GVBD) at 14 and 22 h. At 6 h, only P4 and 17,20-P induced maturation at the highest concentration (1024 ng ml^–1). At 14 and 22 h, 11-deoxycortisol was the most potent steroid inducer of GVBD followed by P4, 17OHP, 17,20-P, and 20-S. The steroid 11-hydroxytestosterone (11OHT) was completely ineffective at all concentrations and exposure times. The C21 steroids induced oocyte maturation at concentrations ranging from 4 to 1024 ng ml^–1, whereas T induced GVBD at 225 to 1139 ng ml^–1. Calculation of the mean effective concentration that induced 50% GVBD (EC50) from the 22 h incubations revealed the following order of potencies: S > P4 > 17OHP > 17,20-P > 20-S >> F > T. These bioassay results, together with previous findings on the endogenous production of steroids by ovarian follicles from gonadotropin-primed females, indicate that more than one steroid has a biological role in the resumption of meiosis in sturgeon oocytes and provides empirical evidence for P4, 17OHP, S, 20-S, and 17,20-P as maturation-inducing steroids in white sturgeon.     

Life-history variation in relation to nest site abundance in males of the freshwater goby Tridentiger brevispinis

Abstract –  Age at maturity in males of the freshwater goby Tridentiger brevispinis , a species with exclusive paternal care, was investigated in two populations in Lake Biwa, Japan, that differed markedly in nest site abundance. At Ohmi-maiko, where nest sites were scarce, most males matured at age 3, and mean body size of males guarding eggs in nests was larger than that of males sampled randomly (including both guarding and nonguarding males) in the population. Conversely, at Minamihama, where nest sites were abundant, many males matured at age 1, and there was no difference in body size between guarding males and males collected randomly. The slope of regression lines between body size of the guarding male and the number of eggs in his nest was greater in Ohmi-maiko than in Minamihama. These results suggest that the shortage of nest sites enhances reproductive success in larger males, probably through male–male competition for nest sites and female mate choice for larger males. I conclude that in nest spawners, the availability of nest sites should strongly affect life-history traits of males through sexual selection.     

猪卵丘卵母细胞复合体和裸卵中卵母细胞发育及基因表达差异的研究

 【目的】研究探讨卵丘细胞的完整性在支持和促进猪卵母细胞成熟过程中发挥的重要作用以及卵丘与卵母细胞互作的分子机理。【方法】对具有完整致密卵丘细胞包裹的和无卵丘细胞包裹的猪卵母细胞进行了体外成熟培养试验,应用mRNA差异显示（DDRT-PCR）和半定量反转录PCR技术(RT-PCR),对这2类猪卵母细胞中mRNA的差异表达进行研究,得到了14条差异表达条带,经过回收、克隆、测序后,再运用半定量（RT-PCR）进行检测。【结果】体外培养的裸卵细胞的各项成熟指标均低于卵丘-卵母细胞复合体。3个基因（PELP1,Myo5b and CAST）和一条新EST序列在有卵丘和无卵丘的猪卵母细胞中表现出差异表达。分离得到的差异表达基因在雌激素受体调节、细胞膜间的信息传导和细胞器物质运输等过程中发挥重要的作用。【结论】这些差异基因可能参与了卵丘细胞与卵母细胞间的相互作用,对卵母细胞的成熟具有重要作用。     

Early metaphase II oocytes treated with dibutyryl cyclic adenosine monophosphate provide suitable recipient cytoplasm for the production of miniature pig somatic cell nuclear transfer embryos

We investigated the effects of in vitro maturation duration and treatment with dibutyryl cyclic adenosine monophosphate (dbcAMP) on the blind enucleation efficiency and developmental competence of miniature pig somatic cell nuclear transfer (SCNT) embryos. Oocytes were cultured for 22 h in NCSU-23 medium with or without 1 mM dbcAMP and then additionally cultured in dbcAMP-free NCSU-23 for 14, 18, or 22 h. Regardless of dbcAMP treatment, the rate of nuclear maturation reached a plateau at 36 and 40 h. However, mitochondrial distribution, a marker for cytoplasmic maturation, differed between the dbcAMP-untreated oocytes at 36 h and dbcAMP-treated oocytes at 40 h. The metaphase II chromosomes were adjacent to the first polar body in 68.8% and 63.5% of the dbcAMP-untreated oocytes at 36 h and dbcAMP-treated oocytes at 40 h, respectively. Furthermore, the blind enucleation efficiency by removing a small volume of cytoplasm was significantly higher in the dbcAMP-untreated oocytes at 36 h (82.9%) and dbcAMP-treated oocytes at 40 h (89.9%) than other groups. The rate of blastocyst formation was highest in the dbcAMP-treated oocytes at 40 h. Hence, this study demonstrated that dbcAMP-treated early metaphase II oocytes are suitable for the production of miniature pig SCNT embryos.