Postpartum fatty liver in high-producing dairy cows in practice and in animal studies. The connection with health, production and reproduction problems

Hepatic lipidosis postpartum (pp) in high-producing dairy cows is associated with an increased vulnerability to infectious diseases, an increased frequency of metabolic diseases and substantial reproduction problems. To enable research that aims at elucidation of the relationship between the hepatic lipidosis postpartum on one hand and the aforementioned problems on the other, an animal model for hepatic lipidosis was developed. Using this model, evidence was found that the sudden and substantial increase of hepatic uptake of free fatty acids contributes to accumulation of triacyglycerol in the liver. As far as the increased susceptibility for infectious diseases is concerned, results were obtained that indicate that in cows with hepatic lipidosis, the immune response is suppressed. With respect to the fertility problems, with the help of in vitro tests, it was found that the quality of oocytes collected in cows with hepatic lipidosis 80–140 days postpartum was decreased. Presented at the ISACB Conference, Glasgow, July 1996.     

激光扫描共聚焦显微镜观察小鼠MⅡ期卵母细胞纺缍体和染色体的实验研究

目的以小鼠为研究模型,探索观察成熟卵母细胞纺缍体和染色体的方法.方法运用激光扫描共聚焦显微镜(LSCM)的光学切片和三维图像重建技术,结合悬浮细胞的免疫化学方法,系统观察了小鼠MⅡ期卵母细胞的纺缍体和染色体.结果应用LSCM清晰地观察到了卵母细胞纺缍体和染色体的形态.发现小鼠成熟卵母细胞纺缍体和染色体的正常率分别为82%和86%.结论 LSCM能够清晰有效地观察MⅡ期卵母细胞的纺缍体和染色体,并可作为评价成熟卵母细胞质量的可靠手段.     

Vitrification of embryos and oocytes for fertility preservation in cancer patients

As survival rates and the life expectancy of those with malignancy have increased, more women in their reproductive years are referred for fertility preservation. Chemotherapy and radiotherapy can severely affect ovarian function, and the effect is irreversible. Therefore, it is optimal to attempt fertility preservation before chemotherapy and radiotherapy are initiated. Oocyte and embryo cryopreservation is the most common option for fertility preservation in women. Several reports have proven that embryo and oocyte cryopreservation can achieve a successful pregnancy. This review discusses the impact of chemotherapy and radiotherapy on ovarian function, and the importance of oocyte and embryo cryopreservation for fertility preservation. In addition, the current status of pregnancy outcomes and potential for cryopreserved oocytes to result in live births in cancer patients was reviewed. This may provide useful information for decision‐making in cancer patients regarding oocyte and embryo cryopreservation and fertility preservation.     

Roles of epidermal growth factor (EGF)-like factor in the ovulation process

Luteinizing hormone (LH) surge stimulates preovulatory follicles to induce the ovulation process, including oocyte maturation, cumulus expansion, and granulosa cell luteinization. The matured oocytes surrounded by an expanded cumulus cell layer are released from follicles to the oviduct. However, LH receptors are dominantly expressed in granulosa cells, but less in cumulus cells and are not expressed in oocytes, indicating that the secondary factors expressed and secreted from LH-stimulated granulosa cells are required for the induction of the ovulation process. Prostaglandin and progesterone are well-known factors that are produced in granulosa cells and then stimulate in both granulosa and cumulus cells. The mutant mice of prostaglandin synthase (Ptgs2KO mice) or progesterone receptor (PRKO mice) revealed that the functions were essential to accomplish the ovulation process, but not to induce the ovulation process. To identify the factors initiating the transfer of the stimuli of LH surge from granulosa cells to cumulus cells, M. Conti’s lab and our group performed microarray analysis of granulosa cells and identified the epidermal growth factor (EGF)-like factor, amphiregulin (AREG), epiregulin (EREG), and β-cellulin (BTC) that act on EGF receptor (EGFR) and then induce the ERK1/2 and Ca²⁺-PLC pathways in cumulus cells. When each of the pathways was down-regulated using a pharmacological approach or gene targeting study, the induction of cumulus expansion and oocyte maturation were dramatically suppressed, indicating that both pathways are inducers of the ovulation process. However, an in vitro culture study also revealed that the EGFR-induced unphysiological activation of PKC in cumulus cells accelerated oocyte maturation with low cytostatic activity. Thus, the matured oocytes are not arrested at the metaphase II (MII) stage and then spontaneously form pronuclei. The expression of another type of EGF-like factor, neuregulin 1 (NRG1), that does not act on EGFR, but selectively binds to ErbB3 is observed in granulosa cells after the LH surge. NRG1 supports EGFR-induced ERK1/2 phosphorylation, but reduces PKC activity to physiological level in the cumulus cells, which delays the timing of meiotic maturation of oocytes to adjust the timing of ovulation. Thus, both types of EGF-like factor are rapidly induced by LH surge and then stimulate cumulus cells to control ERK1/2 and PKC pathways, which results in the release of matured oocytes with a fertilization competence.     

线粒体功能对辅助生殖技术的影响

卵母细胞质量与不孕、发育异常、囊胚数目的减少以及胚胎丢弃有关,而线粒体的功能是影响卵母细胞质量的重要因素,同时也是受精和获得健康后代的重要决定因素。越来越多的女性不孕症患者伴随着诸如糖尿病和肥胖等代谢疾病以及卵母细胞老化等问题,这类人群因为线粒体功能异常,在进行辅助生殖技术助孕的过程中往往也较难取得令人满意的妊娠结局。为了修复卵母细胞质量提高辅助生殖技术的成功率,核置换、极体移植等新技术引起了人们的重视。     

Electrical activation of rabbit oocytes increases fertilization and embryo development by intracytoplasmic sperm injection using sperm from deceased male

Purpose

We investigated the effect of electrical stimulation on rabbit oocyte activation using intracytoplasmic sperm injection (ICSI) to determine whether viable offspring can be produced from deceased rabbit sperm using ICSI.

Methods

Sperm were collected from a heterozygote GFP male rabbit 5 h after sacrifice and cryopreserved in liquid nitrogen. Mature oocytes were fertilized using ICSI. A series of electrical pulse procedures were used to activate oocytes before and/or after ICSI. Following ICSI, zygotes were cultured in B₂ medium for 4 days or transferred into the oviducts of recipient rabbits at the 2- or 4-cell stage.

Results

The blastocyst formation rate was significantly greater in oocytes that received one or two pulses prior to ICSI compared to controls and other electrically stimulated groups. In the single pulse before ICSI group, 23 % of the blastocysts expressed GFP, which was significantly greater than all other groups. However, those that received treatment before and after, or just following ICSI, showed a significant decrease in embryo survival. Finally, embryos from the single pulse before ICSI group were transferred into recipient female rabbits and a full-term kit was successfully delivered.

Conclusions

One pulse of electrical stimulation prior to sperm injection was an effective method to activate rabbit oocytes for fertilization. Sperm collected from a deceased rabbit is able to produce viable embryos through ISCI that are capable of normal fetal and kit development.     

肥胖引起的线粒体功能及抗氧化异常促进小鼠卵母细胞排卵后老化

目的: 探讨肥胖对小鼠卵母细胞体内老化、体外受精和胚胎发育的影响及其机制。方法: 分别获取人绒毛膜促性腺激素(HCG)注射后14 h、18 h和22 h的卵母细胞-卵丘颗粒细胞复合体,进行体外受精胚胎培养,采用Hoechst 33342和碘化丙啶双染检测卵丘颗粒细胞的凋亡率,JC-1和DCFH-DA分别检测卵母细胞中线粒体膜电位(MMP)和活性氧类(ROS)水平,并检测卵母细胞中ATP和谷胱甘肽(GSH)水平。结果: 刚排卵时肥胖小鼠卵母细胞GSH低于对照组(P<0.05),囊胚形成率、ATP、ROS和卵丘颗粒细胞凋亡率两组无显著差别(P>0.05);HCG注射后18 h,肥胖小鼠卵母细胞中ATP含量及囊胚细胞数目开始明显减少并低于对照组(P<0.05),而卵母细胞中ROS水平和卵丘颗粒细胞凋亡率则开始升高并高于对照组(P<0.05);HCG注射后22 h,肥胖小鼠卵母细胞MMP和体外受精囊胚形成率显著下降(P<0.05),并且低于对照组(P<0.05)。结论: 肥胖促进排卵后卵母细胞的老化,氧自由基对卵母细胞的损伤可能是引起母源老化不孕的重要原因。     

尾吊模型模拟微重力抑制小鼠卵母细胞的成熟

本文研究模拟微重力对小鼠卵母细胞成熟的影响,探讨尾吊模型模拟微重力研究雌性小鼠生殖功能的可行性。采用转壁式生物反应器模拟微重力体外培养小鼠卵母细胞,以及对小鼠进行旋转和尾吊处理,观察小鼠卵母细胞的成熟情况。结果表明:体外旋转培养和尾吊模型产生的模拟微重力条件下,小鼠卵母细胞的成熟受到抑制,异常率增加。表现为体外旋转培养小鼠卵母细胞的成熟率为8.93%,显著低于1g重力条件下的72.33%;尾吊模型小鼠卵母细胞的成熟率为14.54%,显著低于对照组的73.29%。与对照组比较,小鼠旋转模型对小鼠卵母细胞的成熟无明显影响,提示该旋转模型不适用于模拟微重力的研究。尾吊模型可适用于地面研究模拟微重力对母鼠繁殖机能的影响。