微流控芯片加载低温保护剂过程中卵母细胞的损伤评估

卵母细胞的低温保存为辅助生殖技术和胚胎工程技术提供了更大的发展空间,而低温保存需要添加高浓度保护剂,会对细胞造成渗透损伤及毒性损伤.与分步法添加固定浓度的保护剂不同,微流控法能够实现保护剂浓度的连续性变化,关于微流控法连续性添加保护剂时卵母细胞的损伤评估还未见报道.本文首先采用数值模拟的方法,模拟细胞在不同加载时间、不同加载线型(线性、S型、凹型)、不同凹型加载(低凹型、高凹型)中细胞的渗透行为,计算各方案中细胞的传统的损伤评估参数:体积变化极值(ΔV)、积累性渗透损伤值(AOD)及毒性损伤值(J).在此基础上,藉由信息熵理论首次提出了综合损伤评估参数s,并通过猪卵母细胞微流控加载后孤雌激活实验的结果验证评估效果.结果表明,对于不同的加载方案,传统的损伤评估参数结果之间出现分歧,无法得到统一的结论.通过分析囊胚率同综合损伤评估参数s值的关系,发现二者呈负相关关系,且相关系数很高,说明综合损伤评估参数s能够较好地对细胞损伤进行评估,为细胞损伤评估开辟了新思路.     

From global proteome profiling to single targeted molecules of follicular fluid and oocyte: contribution to embryo development and IVF outcome

The development of in vitro fertilization (IVF) techniques for infertility management has led to the investigation of the proteome of follicular fluid and oocyte. In addition, different markers contributing to oocyte maturation and embryo development potential have been reported in the literature. Different techniques were utilized to analyze whole proteome or single protein markers in follicular fluid and oocytes, particularly in animal models. Data from several studies have generated large amounts of information, however, an ideal profile to predict the best oocytes and embryos suitable for implantation are still to be uncovered. The identification of such profiles and markers from follicular fluid, oocytes and endometrium should help scientists and clinicians develop better strategies to improving clinical outcome of IVF cycles.     

Culture and Co-Culture of Mouse Ovaries and Ovarian Follicles

The mammalian ovary is composed of ovarian follicles, each follicle consisting of a single oocyte surrounded by somatic granulosa cells, enclosed together within a basement membrane. A finite pool of follicles is laid down during embryonic development, when oocytes in meiotic arrest form a close association with flattened granulosa cells, forming primordial follicles. By or shortly after birth, mammalian ovaries contain their lifetime’s supply of primordial follicles, from which point onwards there is a steady release of follicles into the growing follicular pool.The ovary is particularly amenable to development in vitro, with follicles growing in a highly physiological manner in culture. This work describes the culture of whole neonatal ovaries containing primordial follicles, and the culture of individual ovarian follicles, a method which can support the development of follicles from an immature through to the preovulatory stage, after which their oocytes are able to undergo fertilization in vitro. The work outlined here uses culture systems to determine how the ovary is affected by exposure to external compounds. We also describe a co-culture system, which allows investigation of the interactions that occur between growing follicles and the non-growing pool of primordial follicles.     

Phosphoproteomics of the goat milk fat globule membrane: New insights into lipid droplet secretion from the mammary epithelial cell

Mechanisms of milk lipid secretion are highly controversial. Analyzing the fine protein composition of the “milk fat globule membrane” (MFGM), the triple‐layered membrane surrounding milk lipid droplets (LDs) can provide mechanistic clues to better understand LD biosynthesis and secretion pathways in mammary epithelial cells (MECs). We therefore combined a high‐sensitive Q‐Exactive LC‐MS/MS analysis of MFGM‐derived peptides to the use of an in‐house database intended to improve protein identification in the goat species. Using this approach, we performed the identification of 442 functional groups of proteins in the MFGM from goat milk. To get a more dynamic view of intracellular mechanisms driving LD dynamics in the MECs, we decided to investigate for the first time whether MFGM proteins were phosphorylated. MFGM proteins were sequentially digested by lysine‐C and trypsin proteases and the resulting peptides were fractionated by a strong cation exchange chromatography. Titanium beads were used to enrich phosphopeptides from strong cation exchange chromatography eluted fractions. This approach lets us pinpoint 271 sites of phosphorylation on 124 unique goat MFGM proteins. Enriched GO terms associated with phosphorylated MFGM proteins were protein transport and actin cytoskeleton organization. Gained data are discussed with regard to lipid secretory mechanisms in the MECs. All MS data have been deposited in the ProteomeXchange with identifier PXD001039 ( http://proteomecentral.proteomexchange.org/dataset/PXD001039 ).     

Effect of substituting different concentrations of soybean lecithin and egg yolk in tris-based extender on goat semen cryopreservation

This study aimed to investigate the effects of different concentrations of soybean lecithin (SL; 0.5%, 1%, and 1.5%) and egg yolk (EY) in Tris-based extenders on the semen quality parameters of post-thawed goat semen. Sixteen ejaculates were collected from eight healthy, mature Chongming White goats (3–5 years of age). Each ejaculate was divided into five equal aliquots, and then each pellet was diluted with one of the five Tris-based extenders containing 20% EY, 0.5% SL, 1% SL, 2% SL, or 3% SL. The cooled diluted semen was loaded into 0.5 mL polyvinyl French straws and cryopreserved in liquid nitrogen. Frozen semen samples were thawed at 37 °C and assessed for sperm motility, viability, plasma acrosome integrity, membrane integrity, and mitochondria integrity, and the spermatozoa were assessed for reactive oxygen species (ROS), superoxide dismutase (SOD), and malondialdehyde (MDA). The semen extended in the 2.0% SL extract tended to have a higher sperm viability (57.44%), motility (52.14%), membrane integrity (45.31%), acrosome integrity (52.96%), and mitochondrial activity (50.21%) than the other SL-based extender concentrations (P < 0.05). The 2.0% SL treatment group was equivalent to the semen extended in 20% EY (P > 0.05). The extenders supplemented 20% EY or 2.0% SL significantly increased the SOD activity and decreased the ROS and MDA activities compared to the other groups (P < 0.05). In conclusion, the extenders supplemented with 20% EY and 2.0% SL had similar effects on spermatozoa preservation. These results indicate that a soybean lecithin-based diluent may be used as an alternative extender to egg yolk for the cryopreservation of goat semen.     

Mouse strain‐dependent egg factors regulate calcium signals at fertilization

Calcium (Ca²⁺) signals triggered at fertilization initiate resumption of the cell cycle and initial steps of embryonic development. In mammals, the sperm factor phospholipase Cζ triggers the release of Ca²⁺ from the endoplasmic reticulum (ER), initiating an oscillatory pattern of Ca²⁺ transients that is modulated by egg factors including Ca²⁺ influx channels, Ca²⁺ transporters, and phosphoinositide‐regulating enzymes. Here we compared characteristics of Ca²⁺ oscillations following in vitro fertilization (IVF) and ER Ca²⁺ stores among nine common laboratory mouse strains: CF1, C57BL6, SJL, CD1, DBA, FVB, 129X1, BALBc, 129S1, and the F1 hybrid B6129SF1. Sperm from B6SJLF1/J males was used for all IVF experiments. There were significant differences among the strains with respect to duration and maximum amplitude of the first Ca²⁺ transient, frequency of oscillations, and ER Ca²⁺ stores. With male strain held constant, the differences in Ca²⁺ oscillation patterns observed result from variation in egg factors across different mouse strains. Our results support the importance of egg‐intrinsic properties in determining Ca²⁺ oscillation patterns and have important implications for the interpretation and comparison of studies on Ca²⁺ dynamics at fertilization.     

DNA methylation establishment of CpG islands near maternally imprinted genes on chromosome 7 during mouse oocyte growth

The genome methylation is globally erased in early fetal germ cells, and it is gradually re‐established during gametogenesis. The expression of some imprinted genes is regulated by the methylation status of CpG islands, while the exact time of DNA methylation establishment near maternal imprinted genes during oocyte growth is not well known. Here, growing oocytes were divided into three groups based on follicle diameters including the S‐group (60–100 μm), M‐group (100–140 μm), and L‐group (140–180 μm). The fully grown germinal vesicle (GV)‐stage and metaphase II (M2)‐stage mature oocytes were also collected. These oocytes were used for single‐cell bisulfite sequencing to detect the methylation status of CpG islands near imprinted genes on chromosome 7. The results showed that the CpG islands near Ndn, Magel2, Mkrn3, Peg12, and Igf2 were completely unmethylated, but those of Peg3, Snrpn, and Kcnq1ot1 were hypermethylated in MII‐stage oocytes. The methylation of CpG islands near different maternal imprinted genes occurred asynchronously, being completed in later‐stage growing oocytes, fully grown GV oocytes, and mature MII‐stage oocytes, respectively. These results show that CpG islands near some maternally imprinted genes are not necessarily methylated, and that the establishment of methylation of other maternally imprinted genes is completed at different stages of oocyte growth, providing a novel understanding of the establishment of maternally imprinted genes in oocytes.     

Vitrification of caprine secondary and early antral follicles as a perspective to preserve fertility function

The present study aimed to evaluate the structure, survival and development of isolated caprine (secondary-SEC and early antral-EANT) follicles, after vitrification in the presence of synthetic polymers and in vitro culture. Additionally, transzonal projections (TZPs) and p450 aromatase enzyme were evaluated. After isolation, SEC and EANT follicles were in vitro cultured for six days or vitrified. After one week, SEC and EANT follicles were warmed and also in vitro cultured for six days. Data revealed that the percentage of morphologically normal follicles was similar between fresh and vitrified follicles in both follicular categories and antrum formation rate was similar between fresh and vitrified SEC follicles. Fluorescence by calcein-AM did not show difference between fresh and vitrified (SEC and EANT) follicles, however, the trypan blue test showed low viability for vitrified follicles. The integrity of TZPs was not affected between fresh and vitrified SEC follicles, however, in vitrified EANT follicles, there were signs of TZPs loss. Regarding steroidogenic function, it was observed a positive staining for p450 aromatase enzyme in fresh and vitrified SEC and EANT follicles. It was concluded that SEC follicles seem to be more resistant to vitrification than EANT follicles, as shown by the trypan blue test and TZPs assay. Future studies may confirm this hypothesis, in order to consolidate the use of SEC and EANT follicles as an alternative to ovary cryopreservation.