卵泡液蛋白质组学研究进展

蛋白质组的概念最初在1994年提出.蛋白质组学是以细胞内全部蛋白质的存在及其活动为研究对象,它极大地促进了我们对后基因组时代基因功能的理解.卵泡液作为卵母细胞生长和分化的培养基,直接或间接影响卵母细胞的生育力和发育潜能,其中的一些蛋白质可以作为卵母细胞成熟的标记.应用蛋白质组学方法可以筛选与卵母细胞成熟及一些与生殖疾病有关的标记蛋白.本文就卵泡液的蛋白质组学研究进展作简要综述.     

小鼠腔前卵泡体外培养中BMP-6和GDF-9的表达

目的观察BMP-6及GDF-9蛋白在小鼠体外培养卵泡中的定位和定量表达,探讨二者与卵泡发育的关系。方法采用免疫荧光和western blot技术观察BMP-6及GDF-9在体外培养的第6、10天卵泡的定位和定量表达情况。结果在体外培养中,腔前卵泡和有腔卵泡的卵母细胞和颗粒细胞中均检测到BMP-6和GDF-9蛋白的表达;western blot定量显示,在卵泡体外发育的不同阶段,BMP-6和GDF-9蛋白的表达水平不同。结论 BMP-6及GDF-9蛋白存在于体外培养卵泡的卵母细胞和颗粒细胞中;二者的表达水平随卵泡的发育成熟而发生变化。     

卵泡内环境对猪卵泡卵体外成熟和发育的影响

研究卵泡内环境对猪卵母细胞体外成熟、受精及受精卵体外发育的影响。主要结果如下：直径≥5mm、4－4.9mm、3－3.9mm和2－2.9mm的卵泡卵母细胞体外成熟率分别为90.5%、89.7%、85.4%和67.4%,体外受精后,卵母细胞的发育能力随卵泡直径的增大而增强,直径≥5mm和4－4.9mm卵泡卵的2－细胞、3－4－细胞发育率显著高于直径2－2.9mm的卵泡卵（P＜0.05或0.01）。体外成熟培养36h、42h和48h,直径2－2.9mm卵泡卵的体外成熟率,体外受精后的卵裂率差异不显著（P＞0.05）。在体外成熟培养液中添加5％或15％的不同直径卵泡的卵泡液,各组间卵母细胞的体外成熟率,受精卵的体外发育率均无显著差异,结果表明：卵泡大小对猪卵母细胞体外成熟、受精及受精卵体外发育有重要影响。     

卵泡液蛋白质组学2-DE体系的构建与应用

卵泡液是在卵泡形成过程中生成的,作为卵母细胞生长和分化的培养基,直接或间接影响卵母细胞的生育力和发育潜能,其成分非常复杂。本研究通过调整,优化2-DE技术条件,建立了相对稳定的卵泡液蛋白质2-DE技术,获得了卵泡液蛋白质组表达图谱,并用PDQuest软件进行了图像分析,用MALDI-TOF MS鉴定了9个蛋白点,其中有四种为新鉴定的在卵泡液中表达的蛋白质。结果表明,该体系稳定性、重复性好,为进一步开展卵泡液蛋白质组学的相关研究奠定了基础。     

猪卵泡内环境对其卵母细胞体外成熟与发育的影响

目的：研究猪卵泡内环境对卵母细胞体外成熟,受精及受精卵体外发育的影响。结果：直径≥5mm,4-4.9mm,3-3.9mm,2-2.9mm的卵泡内卵母细胞体外成熟率分别为90．5％,89．7％,85．4％和67．4％。体外受精后,受精卵的发育能力随卵泡直径的增大而增强,来自直径≥5mm和4－4．9mm卵泡的受精卵发育到2－细胞,3－4细胞的比率显高于来自直径2－2．9mm的卵泡受精卵（P＜0．05或0．01）,体外成熟培养36,42,48小时,直径2－2．9mm卵泡卵的体外成熟率,体外受精后的卵裂率差异不显（P＞0．05）。在体外成熟培养液中添加5％或15％的不同直径卵泡的卵泡液,各组间卵母细胞的体外成熟率,受精卵的体外发育率均无显差异。结论：卵泡大小对猪卵母细胞体外成熟,受精及受精卵体外发育有重要影响。     

卵泡液中细胞外囊泡及其携带的microRNA对卵泡发育的作用

细胞外囊泡(Extracellular vesicles,EVs)是指细胞分泌的双层膜转运囊泡。EVs能从细胞中摄取大分子物质,并将其转移至受体细胞。在这些大分子物质中,研究最多的就是microRNA (miRNA)。miRNA是一种参与基因表达调控的非编码RNA,已证实在哺乳动物卵泡液EVs中有不同的非编码RNA存在,EVs携带miRNA可以作为自分泌和旁分泌的替代机制,影响卵泡发育。文中系统介绍了EVs的种类、特征和分离鉴定方法,重点综述了EVs及携带的miRNA对卵泡发育的作用,包括早期卵泡发育、卵母细胞成熟、卵泡优势化以及对颗粒细胞功能的影响。同时对卵泡液中EVs及其携带的miRNA的未来研究进行了展望,为卵泡液中EVs及携带的miRNA功能的研究及应用提供了思路和方向。     

干细胞因子在卵泡发育过程中的作用

干细胞因子(stemcellfactor,SCF)是酪氨酸激酶受体的配体。哺乳动物卵巢组织能表达SCF,它不仅能促进和诱导卵母细胞的发育,并能调控卵泡细胞间的相互作用及激素的生成,而且是卵泡发育过程中重要的旁分泌因子,可能激活蛋白激酶C(PKC)和有丝分裂原激活蛋白激酶的激酶(MEK)及PI3激酶途径信号分子等信号途径,对卵泡发育起调节作用。     

卵泡液细胞外囊泡携带的microRNA对卵泡闭锁影响的研究进展

细胞外囊泡(extracellular vesicles, EVs)是细胞主动释放的膜结合颗粒。在原核生物和真核生物中,EVs被认为是细胞间进行信息交流的一种方式。EVs具有携带蛋白质、脂质和核酸等生物大分子的能力,可以影响亲本细胞和受体细胞的不同生理功能。其中,EVs携带的microRNA研究报道最多,在生物体生理功能方面发挥着重要作用。卵泡在发育过程中,只有少数卵泡可以充分发育、成熟、排卵,大多数卵泡在发育的不同阶段发生闭锁。在卵泡发育的整个过程中,每一阶段的变化以及卵泡闭锁调控机制还不完全清楚。本文在总结EVs类型、特性、分离方法及用途的基础上,从不同细胞因子、激素方面重点论述了卵泡液中EVs携带的microRNA是如何调控卵泡闭锁,同时对卵泡液EVs携带的microRNA在生殖调控和生殖疾病诊断方面的研究前景进行了展望,对于卵泡发育调控研究以及有效利用研究具有一定参考意义。     

新生小鼠卵巢移植雄鼠肾囊下卵泡的生长发育

将1日龄小鼠卵巢移植入成年雄鼠肾囊下,分别于移植后18d、36d回收移植卵巢进行形态学、组织学观察,以评价卵巢移植体在成年雄性受体小鼠体内生长及卵泡发育潜能。结果表明:移植体生长增大,有各级生长卵泡发育;18日龄移植体平均直径为1881.1μm±204.7μm,与1日龄卵巢相比差异极显著(P<0.01),卵泡发育到有腔卵泡阶段;36日龄移植体平均直径达2575.3μm±466.4μm,显著大于18日龄移植体(P<0.01),有成熟卵泡出现,未观察到黄体;从移植体分离到GV期卵母细胞和卵丘卵母细胞复合体。研究表明1日龄小鼠卵巢移植体在雄性受体生理环境中具有正常生长发育和形成成熟卵泡的潜能。     

卵泡颗粒细胞凋亡相关蛋白的研究进展

哺乳动物的卵巢中存在大量卵泡。大多数卵泡在发育过程中发生闭锁而消失,只有少数可以发育到成熟而排卵。卵泡是由卵母细胞与其周围的颗粒细胞构成的。卵泡颗粒细胞的凋亡是卵泡闭锁的主要原因。颗粒细胞凋亡相关蛋白通过参与凋亡通路及凋亡信号转导调节凋亡。本文就哺乳动物卵泡颗粒细胞凋亡相关蛋白的研究进展进行综述。     

Proteomic analysis of human follicular fluid from fertile women

Background

Follicular fluid is a unique biological fluid in which the critical events of oocyte and follicular maturation and somatic cell-germ cell communication occur. Because of the intimate proximity of follicular fluid to the maturing oocyte, this fluid provides a unique window into the processes occurring during follicular maturation. A thorough identification of the specific components within follicular fluid may provide a better understanding of intrafollicular signaling, as well as reveal potential biomarkers of oocyte health for women undergoing assisted reproductive treatment. In this study, we used high and low pH HPLC peptide separations followed by mass spectrometry to perform a comprehensive proteomic analysis of human follicular fluid from healthy ovum donors. Next, using samples from a second set of patients, an isobaric mass tagging strategy for quantitative analysis was used to identify proteins with altered abundances after hCG treatment.

Results

A total of 742 follicular fluid proteins were identified in healthy ovum donors, including 413 that have not been previously reported. The proteins belong to diverse functional groups including insulin growth factor and insulin growth factor binding protein families, growth factor and related proteins, receptor signaling, defense/immunity, anti-apoptotic proteins, matrix metalloprotease related proteins, and complement activity. In a quantitative analysis, follicular fluid samples from age-matched women undergoing in vitro fertilization oocyte retrieval were compared and 17 follicular fluid proteins were found at significantly altered levels (p < 0.05) between pre-hCG and post-hCG samples. These proteins belong to a variety of functional processes, including protease inhibition, inflammation, and cell adhesion.

Conclusions

This database of FF proteins significantly extends the known protein components present during the peri-ovulatory period and provides a useful basis for future studies comparing follicular fluid proteomes in various fertility, disease, and environmental exposure conditions. We identified 17 differentially expressed proteins after hCG treatment and together these data showed the feasibility for defining biomarkers that illuminate how the ovarian follicle microenvironment is altered in various infertility-related conditions.

Electronic supplementary material

The online version of this article (doi:10.1186/s12014-015-9077-6) contains supplementary material, which is available to authorized users.     

Proteomic analysis of recurrent spontaneous abortion: Identification of an inadequately expressed set of proteins in human follicular fluid

Recurrent spontaneous abortion (RSA), defined as the loss of three or more consecutive pregnancies prior to the 20th week of gestation, affects up to 5% of the child-bearing population. To investigate the proteins associated with RSA, the protein expression in human follicular fluid was analyzed using 2-DE. Follicular fluid contains a variety of biologically important proteins for oocyte fertilization and follicle maturation in the mammalian reproductive process. Therefore, it can be used as a provisional source for identifying proteins involved in RSA. In this study, we identified five aberrantly expressed proteins (complement component C3c chain E, fibrinogen gamma, antithrombin, angiotensinogen, and hemopexin precursor) in follicular fluid from RSA patients with MALDI-TOF-MS and nano-LC MS/MS. Western blot analysis confirmed that the protein expression level of fibrinogen gamma and antithrombin was less in follicular fluid from RSA patients than those from normal controls. Semiquantitative RT-PCR and real-time PCR analyses revealed that mRNA level of these coagulation factors was also decreased significantly in chorionic villi of RSA patients compared with normal samples. Taken all together, it is likely that coagulation factors (fibrinogen gamma and antithrombin) play an important role in maintaining the normal pregnancy.     

S100‐A9 protein in exosomes derived from follicular fluid promotes inflammation via activation of NF‐κB pathway in polycystic ovary syndrome

Exosomes have recently emerged as key mediators of different physiological and pathological processes. However, there has been few report about proteomic analysis of exosomes derived from human follicular fluid and their association with the occurrence of PCOS. Herein, we used TMT‐tagged quantitative proteomic approach to identify proteomic profiles in exosomes derived from follicular fluid of PCOS patients and healthy controls. We identified 662 proteins in exosomes derived from human ovarian follicular fluid. Eighty‐six differently expressed proteins (P < .05) were found between PCOS and healthy women. The alterations in the proteomic profile were related to the inflammation process, reactive oxygen species metabolic process, cell migration and proliferation. Importantly, we observed that follicular fluid exosomes contain S100 calcium‐binding protein A9 (S100‐A9) protein. Exosome‐enriched S100‐A9 significantly enhanced inflammation and disrupted steroidogenesis via activation of nuclear factor kappa B (NF‐κB) signalling pathway. These data demonstrate that exosomal proteins are differentially expressed in follicular fluid during disease process, and some proteins may play important roles in the regulation of granulosa cell function. These results highlight the importance of exosomes as extracellular communicators in ovarian follicular development.     

Proteome analysis of human follicular fluid

We used proteomic approach to analyze the protein profile of human follicular fluid (HFF) obtained from 25 normo-ovulatory women undergoing assisted reproduction techniques due to a male infertility factor. In all HFF samples analyzed we found 695 common spots distributed in the 3 to 10 pH range and in the 10-200 kDa range. Only 625 of these spots were also present in the plasma. We used MALDI-TOF-MS analysis to unequivocally assign 183 HFF/plasma matched spots and 27 HFF/plasma unmatched spots. A large number of acute-phase proteins, including transferrin, ceruloplasmin, afamin, hemopexin, haptoglobin and plasma amyloid protein, were identified in HFF in relatively high concentration supporting the hypothesis that mammalian ovulation can be compared to an inflammatory event. We also identified several important antioxidant enzymes; i.e., catalase, superoxide dismutase, glutathione transferase, paraoxonase, heat shock protein 27 and protein disulfide isomerase. This indicates that during maturation the human follicle is well protected against toxic injury due to oxidative stress.     

In vitro equine oocyte maturation in pure follicular fluid plus interleukin-1 and fertilization following ICSI

The interleukin-1 (IL-1) system is thought to be involved in periovulatory events in the mare. Previous in vivo studies have demonstrated that IL-1β induces oocyte maturation, but depresses the pregnancy rate 14 days after ovulation. To better understand the role of IL-1 in oocyte maturation and fertilization, the effects of IL-1 on the in vitro maturation rate of equine oocytes in pure follicular fluid were evaluated and fertilization rate assessed following intracytoplasmic sperm injection (ICSI). Oocytes collected from slaughterhouse ovaries were cultured in four different media for 30 h prior to fertilization. Two experiments were performed, each using three maturation media as the experimental treatments. Medium 1 was pure follicular fluid from subordinate follicles. Medium 2 was medium 1 plus 50 ng/ml recombinant human IL-1β. Medium 3 was pure follicular fluid collected from mares administered crude equine gonadotropin (CEG). Medium 4 was medium 2 plus 50 ng/ml of recombinant human IL-1 receptor antagonist. Media 1, 2 and 3 were compared in experiment 1. In experiment 2, media 1, 2 and 4 were compared. After maturation, metaphase II oocytes were submitted to microinjection and assessed for signs of fertilization. In experiment 1, 101 oocytes were evaluated. The rate of polar body extrusion was 66, 51 and 68% and the proportions of normally fertilized oocytes after ICSI were 40, 18 and 38% for media 1, 2 and 3, respectively. In experiment 2, 122 oocytes were evaluated. The rate of polar body extrusion was 55, 48 and 42% and the proportions showing normal fertilization after ICSI were 14, 25 and 29% for media 1, 2 and 4, respectively. There was no positive effect of IL-1β on maturation in both experiments, but the fertilization rate and percentage of embryos reaching four-cell were low in the presence of IL-1β, indicating that this cytokine may interfere with fertilization and early embryo development.     

The effects of follicular fluid on in vitro maturation, oocyte fertilization and the development of bovine embryos

We determined the effects of follicular fluid in the maturation medium on bovine oocyte maturation, fertilization and subsequent development, as well as on the number of cells in blastocysts following culture. Fluid and oocytes from bovine follicles less than 5 mm in diameter were collected from the ovaries of slaughtered cows. For the maturation medium, follicular fluid at concentrations of 10, 30 or 60% (v/v) was added to Medium 199 with Earle's salts supplemented with 0.1 microg/ml estradiol-17 beta (E(2), Experiment 1) or 0.1 microg/ml E2 and 100 IU/ml hCG (Experiment 2). The control medium contained polyvinylpyrrolidone (PVP; 3 mg/ml) instead of follicular fluid. After maturation for 24 h, oocytes were fertilized in vitro with bull frozen-thawed spermatozoa and cultured on a monolayer of granulosa cells for 9 d. There were no differences in maturation or fertilization rates of oocytes. In Experiment 1, maturation medium containing 10% follicular fluid did not affect the developmental rate of the oocytes to > 2-cell, 8 to 16-cell, blastocyst and hatched blastocyst stage embryos, respectively; whereas 60% decreased embryonic development (P < 0.05) compared with the control. Blastocysts and hatched blastocysts developed from fertilized oocytes which had been matured in medium containing 10 and 30% follicular fluid/E(2) had more cells than the controls (P < 0.01). In Experiment 2, maturation medium containing 10 or 30% follicular fluid did not affect the development fertilized oocytes to the blastocyst stage compared with the control, but decreased at 60% (P < 0.01). There were no differences in the number of cells from Day 9 blastocysts and hatched blastocysts from fertilized oocytes matured in maturation medium containing follicular fluid and E(2) + hCG. The results of these experiments suggest that the addition of bovine follicular fluid to the maturation medium enhances the cell numbers in blastocysts from bovine follicular oocytes matured in vitro.     

Pure preovulatory follicular fluid promotes in vitro maturation of in vivo aspirated equine oocytes

In the mare, rates of fertilization and development are low in oocytes matured in vitro, and a closer imitation of in vivo conditions during oocyte maturation might be beneficial. The aims of the present study were, therefore, to investigate whether (1) equine oocytes can be matured in vitro in pure equine preovulatory follicular fluid, (2) priming of the follicular fluid donor with crude equine gonadotrophins (CEG) before aspiration of preovulatory follicular fluid promotes the in vitro maturation rate, (3) the in vitro maturation rate differs between oocytes aspirated during estrus and those aspirated again 8 days after the initial follicular aspiration, and (4) high follicular concentrations of meiosis activating sterols (MAS) are beneficial for in vitro maturation of equine oocytes. During estrus, 19 pony mares were treated with 25 mg CEG. After 24 h (Al) and again after 8 days (A2), all follicles >4mm were aspirated and incubated individually for 30 h in the following culture media: standard culture medium (SM), preovulatory follicular fluid collected before CEG containing low MAS concentrations (FF1), preovulatory follicular fluid collected before CEG containing high MAS concentrations (FF2) or preovulatory follicular fluid collected 35 h after administration of CEG containing low MAS concentrations (FF3). Cumulus expansion rate was significantly affected by culture medium. The overall nuclear maturation rate was significantly higher for oocytes collected at A1 (67%) than for oocytes collected at A2 (30%). For oocytes collected at A1, the maturation rates were 71% (FF1), 61% (FF2), 79% (FF3) and 56% (SM). An electrophoretic protein analysis of the culture media revealed the presence of a 200-kDa protein in FF3. The results demonstrate that (1) equine oocytes can be matured during culture in pure equine preovulatory follicular fluid, (2) preovulatory follicular fluid collected after gonadotrophin-priming seems superior in supporting in vitro maturation than standard culture medium, (3) oocytes aspirated 8 days after a previous aspiration are less competent for in vitro maturation than oocytes recovered during the initial aspiration, and (4) the regulation of meiotic resumption during in vitro culture of equine oocytes might be related to the presence of a 200-kDa protein.     

Proteomic analysis of human follicular fluid using an alternative bottom-up approach

Human follicular fluid (hFF) is the in vivo environment of oocytes during follicular maturation in the ovaries. It contains a huge variety of compounds such as, e.g., proteins that might play an important role in follicular development and oocyte growth. Previous proteomic studies on follicular fluid have isolated and already identified a certain number of proteins. Nevertheless, only a small part of proteins present in follicular fluid have been covered so far and a large number have still not been identified. Therefore, the need for new, more resolving, and sensitive approaches in proteome research is evident. We utilized a proteomic setup based on in solution isoelectric focusing (IEF) and reversed-phase nanoliquid chromatography coupled to matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (nano-LC MALDI TOF/TOF MS) for in depth protein analysis of human follicular fluid samples of patients undergoing controlled ovarian hyper stimulation (COH) for in vitro fertilization therapy (IVF). This approach led to the significant identification of 69 proteins, where 32 have not been reported before to be found in human follicular fluid with proteomic methods. Among these findings, at least two relevant compounds essentially involved in hormone secretion regulation during the folliculogenetic process were identified: sex hormone binding globulin (SHBG) and inhibin A (INHA). To confirm these results, both proteins were further validated by immunoassays.