Metabolomic analysis of oviduct fluid on day 3 post-estrus in Holstein heifers

The metabolites in the oviduct fluid (OF) of both oviducts were analyzed by proton nuclear magnetic resonance (¹H-NMR) in Holstein heifers on day 3 after synchronized estrus. Twenty-six metabolites were quantified, among which lactate, glycine and myoinositol were the most abundant. Six metabolites including glycine and myoinositol varied in amount according to the proximity to the corpus luteum. Glucose and histidine were among the most variable metabolites among heifers while threonine and lactate were among the most stable ones, suggesting different mechanisms of homeostasis in the OF.     

Amino acids in cat fallopian tube and follicular fluids

Aminograms of tubal and follicular fluids were obtained using fluids collected by aspiratory puncture from six cats. The amino acids were separated and quantified by high-performance liquid chromatography analysis. The serum of the cats was used as control. The three most prevalent amino acids quantified in cat tubal fluid were glycine, glutamic acid, and taurine. Their mean concentrations were 840 μmol/l (μm), 808 μm and 596 μm, respectively. The three most prevalent amino acids quantified in cat follicular fluid were alanine, glutamine, and taurine. Their mean concentrations were 359 μm, 351 μm, and 258 μm, respectively. This result is consistent with aminograms of tubal fluid previously determined in other mammals. As previously observed in other species and humans, glycine was quantitatively the most abundant and most prevalent free amino acid in cat tubal fluid. The total quantity of amino acids in tubal fluid was similar in cats and other species. However, in contrast with other species studied, hypotaurine was not detected in tubal and follicular fluids of female cats.     

Miniaturized proteomics and peptidomics using capillary liquid separation and high resolution mass spectrometry

Knowledge of the protein and peptide content in a tissue or a body fluid is vital in many areas of medical and biomedical sciences. Information from proteomic and peptidomic studies may reveal alterations in expression due to, e.g., a disease and facilitate the understanding of the pathophysiology and the identification of biological markers. In this minireview, we discuss miniaturized proteomic and peptidomic approaches that have been applied in our laboratory in order to investigate the protein and peptide contents of body fluids (such as plasma, cerebrospinal and amniotic fluid), as well as extracted tissues. The methods involve miniaturized liquid separation, i.e., capillary liquid chromatography and capillary electrophoresis, combined with high resolution mass spectrometry (MS), i.e., Fourier transform ion cyclotron resonance MS. These approaches provide the opportunity to analyze samples of small volumes with high throughput, high sensitivity, good dynamic range and minimal sample handling. Also, the experiments are relatively easy to automate.     

Endoscopic approaches to manage in vitro and in vivo embryo development: Use of the bovine oviduct

The oviduct plays a major part in different reproductive processes providing the microenvironment for numerous steps in early embryogenesis. Consequently, there is a growing demand to perform comparative studies focusing on causal mechanisms related to embryo development within its environment including complex and holistic strategies. However, the routine flushing and transfer procedure of bovine embryos is limited to the morula and blastocyst stage. Additionally, the use of in vitro production of bovine embryos provides access to an extra amount of embryos at various stages. But the quality of these embryos does not reflect the quality of its ex vivo counterparts. For two decades our own studies have focused on use of the oviductal environment of different species to optimize early embryo development for different purposes. The current article briefly highlights some main characteristics of the fallopian tube and reviews the endoscopic approach to access the fallopian tube using the stepwise minimal invasive technique established in different species.     

Optimization for peptide sample preparation for urine peptidomics

Analysis of native or endogenous peptides in biofluids can provide valuable insights into disease mechanisms. Furthermore, the detected peptides may also have utility as potential biomarkers for non-invasive monitoring of human diseases. The non-invasive nature of urine collection and the abundance of peptides in the urine makes analysis by high-throughput ‘peptidomics’ methods , an attractive approach for investigating the pathogenesis of renal disease. However, urine peptidomics methodologies can be problematic with regards to difficulties associated with sample preparation. The urine matrix can provide significant background interference in making the analytical measurements that it hampers both the identification of peptides and the depth of the peptidomics read when utilizing LC-MS based peptidome analysis. We report on a novel adaptation of the standard solid phase extraction (SPE) method to a modified SPE (mSPE) approach for improved peptide yield and analysis sensitivity with LC-MS based peptidomics in terms of time, cost, clogging of the LC-MS column, peptide yield, peptide quality, and number of peptides identified by each method. Expense and time requirements were comparable for both SPE and mSPE, but more interfering contaminants from the urine matrix were evident in the SPE preparations (e.g., clogging of the LC-MS columns, yellowish background coloration of prepared samples due to retained urobilin, lower peptide yields) when compared to the mSPE method. When we compared data from technical replicates of 4 runs, the mSPE method provided significantly improved efficiencies for the preparation of samples from urine (e.g., mSPE peptide identification 82% versus 18% with SPE; p = 8.92E-05). Additionally, peptide identifications, when applying the mSPE method, highlighted the biology of differential activation of urine peptidases during acute renal transplant rejection with distinct laddering of specific peptides, which was obscured for most proteins when utilizing the conventional SPE method. In conclusion, the mSPE method was found to be superior to the conventional, standard SPE method for urine peptide sample preparation when applying LC-MS peptidomics analysis due to the optimized sample clean up that provided improved experimental inference from the confidently identified peptides.     

Culture of bovine embryos in intermediate host oviducts with emphasis on the isolated mouse oviduct

The oviduct provides the optimal environment for the transport of sperm and oocyte at the earliest stages of mammalian embryo development. During the early postfertilization period, several major developmental events occur in the embryo including (i) the first cleavage division, (ii) activation of the embryonic genome, (iii) compaction of the morula, and (iv) formation of the blastocyst. Most of these events are initiated in the oviduct. The absence of assistance from the oviduct may compromise the developmental ability of the cattle embryo under in vitro culture conditions. The oviducts of several mammalian species, including rabbits, cow, sheep (in situ), and mice (organ culture), can sustain early bovine embryos and yield blastocysts of better quality compared with those of culture conditions in vitro, leading to normal pregnancy rates in recipient animals. This review focuses on the use of oviducts in vitro or in vivo as intermediate hosts for postfertilization culture environment of bovine in vitro-produced zygotes with emphasis on the mouse model.     

Diverse range of small peptides associated with high-density lipoprotein

High-density lipoproteins (HDL) were examined as potential carriers of small peptides in plasma. HDL purified by density gradient centrifugation was delipidated and fractionated by size-exclusion chromatography under denaturing conditions. By HPLC and mass spectrometry, more than 100 peptide components were found in the size range from 1000 to 5000 Da. By sequence analysis, peptides were identified as fragments of proteins such as apolipoproteins, fibrinogen, alpha1-proteinase inhibitor, and transthyretin. The results indicate that purified HDL bears a complex range of small peptides. It is unclear whether the peptides have any significant functional role as apolipopeptides, but they may represent a pathway for peptide delivery or scavenging and a significant reservoir of plasma peptides for diagnostic evaluation.     

Urinary Peptidomic Analysis Identifies Potential Biomarkers for Acute Rejection of Renal Transplantation

Introduction  Human urine is a complex matrix of proteins, endogenous peptides, lipids, and metabolites. The level of any or all of these components can reflect the pathophysiological status of an individual especially of the kidney at the time of urine collection. The naturally occurring endogenous urinary peptides which are thought to be the product of several proteolytic and degradation processes may provide clinically useful biomarkers for different renal and systemic diseases. Materials and Methods  To examine if specific differences in the urinary peptidome (<10 kDa) occur at the time of acute renal transplant rejection (AR), we undertook a study of urine samples collected from biopsy-proven AR (n = 10), stable graft function (n = 10), and healthy normal control (n = 10). The peptides (<10 kDa) were extracted and fractionated with high-performance liquid chromatography followed by matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometric (MS) analysis. Results  We identified 54 endogenous peptides, including multiple peptides for Tamm–Horsfall protein (UMOD). A panel of peptides are identified which discriminate renal transplant patients with AR from stable graft. We have shown that liquid chromatography followed by MALDI is a useful tool to identify potential biomarkers, which after verification with larger patient cohort can be used as a non-invasive monitoring tool for renal transplant rejection.     

小鼠的胚胎移植方法

全面而详细地描述了小鼠胚胎移植中所采用的输卵管移植及子宫移植方法,包括作者的改进。介绍了寄母小鼠的选择、配种及孕鼠的编号方法等,具有较强的实用意义。     

Short-term and long-term effects of embryo culture in the surrogate sheep oviduct versus in vitro culture for different domestic species

The culture of early embryos in the surrogate xeno-oviduct was first developed in the early 1950s to allow transport of embryos at long distances. Later, it was applied to the study of culture requirements of the early embryo especially that of bovine origin. In this article, we review the data available on the culture of in vitro-matured and in vitro-fertilized embryos of Bos taurus, Sus scrofa, Equus caballus and Ovis aries in the surrogate sheep oviduct compared with data on in vitro culture in different media. Short-term and long-term cellular and molecular effects are described mainly for the bovine species where more extensive use of this technique has been made. A comparison with in vitro culture in various conditions and species indicate that embryos cultured in the sheep oviduct have close similarities to totally in vivo-derived embryos. The data provided demonstrate that the technique of in vivo culture in the surrogate sheep oviduct is versatile and allows a high rate of embryonic development in all species examined.     

三种胚胎移植技术方法比较

以近交系FVB、DBA／2小鼠作为供体胚小鼠,封闭群CD-1小鼠作为受体小鼠,对实验小鼠胚胎移植的三种方法进行比较,即：对小鼠受精卵1细胞期胚(或2细胞期胚)经由受体小鼠输卵管口移植、经由受体小鼠输卵管壁移植及对小鼠进行子宫移植(8细胞期胚)的方法进行了实验研究,并对各方法适用于使用的动物对象、实验要求、产仔率、优势与不足等做了进一步探讨,为今后的实验动物提供参考依据。结果表明,输卵管壁移植优于输卵管伞口移植和子宫移植。     

Characterization of the Substrate Specificity of Human Carboxypeptidase A4 and Implications for a Role in Extracellular Peptide Processing

CPA4 (carboxypeptidase A4) is a member of the metallocarboxypeptidase family. CPA4 was originally found in a screen of mRNAs up-regulated by sodium butyrate-induced differentiation of cancer cells. Further studies suggested a relation between CPA4 and prostate cancer aggressiveness. In the present study, we determined that CPA4 is secreted from cells as a soluble proenzyme (pro-CPA4) that can be activated by endoproteases, such as trypsin. Three complementary approaches were used to study the substrate specificity of CPA4; kinetic analysis was performed using a new series of chromogenic substrates and some biologically relevant peptides, the cleavage of synthetic peptides was tested individually, and the cleavage of a mixture of >100 mouse brain peptides was examined using a quantitative peptidomics mass spectrometry-based approach. CPA4 was able to cleave hydrophobic C-terminal residues with a preference for Phe, Leu, Ile, Met, Tyr, and Val. However, not all peptides with C-terminal hydrophobic residues were cleaved, indicating the importance of additional residues within the peptide. Aliphatic, aromatic, and basic residues in the P1 position have a positive influence on the cleavage specificity. In contrast, acidic residues, Pro, and Gly have a negative influence in the P1 position. Some of the peptides identified as CPA4 substrates (such as neurotensin, granins, and opioid peptides) have been previously shown to function in cell proliferation and differentiation, potentially explaining the link between CPA4 and cancer aggressiveness. Taken together, these studies suggest that CPA4 functions in neuropeptide processing and regulation in the extracellular environment.     

醮核荔枝胚胎发育相关蛋白质的分离及初步鉴定

通过二维聚丙烯酰胺凝胶电泳以及计算机辅助的图像分析技术,对荔枝开花后40d的正常与败育胚蛋白质图谱进行初步分析。结果表明,100个蛋白质点在表达丰度上有明显差异;选择仅在正常发育胚胎胶上表达的蛋白点15个和仅在败育胚胎胶上表达的蛋白点50个,进行基质辅助激光解吸附电离飞行时间质谱（MALDI—TOFMASS）分析,鉴定出9个与胚发育相关的蛋白,这些蛋白可能参与了胚败育的调节和控制。     

Progressive degradation of serum samples limits proteomic biomarker discovery

Preanalytical variables play a key role in discovery of biomarkers. Although the effect of several preanalytical variables on the mass spectral profiles has been studied extensively, little is known about long-term storage of serum samples. This is important because samples used in case-control or epidemiological studies are usually stored for a long time before analysis. Here we evaluated long-term storage effects on mass spectral peak patterns of serum peptides extracted using weak cation exchange magnetic beads. For this, 20 serum samples stored at −80 °C were divided equally into two groups based on their storage time. We found that intensities of 26 mass spectral peaks significantly varied between these two groups. Intensities of these peaks significantly correlated with storage time. Genetic algorithm-based models generated using these 26 peaks could classify 63 additional samples into these two groups with 100% and 96% accuracy, respectively. We also show that storing samples for 10 months at −80 and −20 °C results in the appearance/disappearance or intensity variation of peaks, some of which were previously reported as disease biomarkers.     

无核荔枝胚胎发育时期蛋白质图谱分析

通过二维聚丙烯酰胺凝胶电泳(2DE)以及计算机辅助的图像分析技术,对荔枝开花后20d的正常与败育胚蛋白质图谱进行了初步分析。结果表明,正常胚总蛋白质斑点数为129,败育胚总蛋白质斑点数为130,其中24个蛋白质点在两种胚中的表达丰度没有明显变化,35个蛋白质点在表达丰度上有明显差异,55%的蛋白则发生了蛋白质缺失、增加以及位置改变等变化。这两种蛋白质组的表达差异说明了胚内蛋白质成分在其败育过程中发生了变化,这些蛋白可能参与了胚败育的调节和控制。     

Fetal development of mouse oocytes and zygotes cryopreserved in a nonconventional freezing medium

This study (1) analyzed fetal development of mouse embryos after oocyte cryopreservation in CJ2, a choline-based medium, (2) examined the effect of culture duration in vitro on subsequent fetal development, and (3) compared survival and fetal development of zygotes frozen in embryo transfer freeze medium (ETFM; sodium-based medium) or CJ2. Unfertilized oocytes and zygotes were cryopreserved using a slow-cooling protocol. After thawing, oocytes were inseminated after drilling a hole in their zona, cultured in vitro either to the two-cell or blastocyst stage, and transferred to the oviducts or uterine horns of recipient mice. In parallel experiments, frozen-thawed zygotes were similarly cultured and transferred. Implantation rates for transferred embryos were high (range 66-88%), regardless of whether they had been frozen as oocytes or zygotes and whether they had been transferred to the oviduct or uterus. However, fetal development was significantly higher when two-cell embryos were transferred. With blastocyst transfer, control embryos implanted and produced a greater proportion of fetuses than did oocytes frozen in CJ2, whereas transfer at the two-cell stage resulted in similar proportions of implantation sites and fetuses. Blastocyst transfer of zygotes cryopreserved in ETFM or CJ2 produced similar fetal development rates (23.6% vs 20.0%), but when frozen-thawed zygotes were transferred at the two-cell stage the fetal development rates were higher in the ETFM group (53.3%) than in the CJ2 group (32.0%). A high proportion (46.7%) of oocytes frozen in CJ2 in a nonprogrammable freezer and plunged at -20 degrees C developed into live offspring. This study shows that in the mouse (1) oocytes frozen in CJ2 can develop into viable fetuses, (2) prolonging culture in vitro has a detrimental effect on embryo transfer outcome, and (3) CJ2 offers no advantage for zygote cryopreservation.     

解脲支原体对兔输卵管黏膜上皮细胞致病性研究

目的：探讨解脲支原体(UU)对兔输卵管黏膜上皮细胞的粘附及其致病性。方法：采用雌性大白兔作为实验对象,用UU感染兔输卵管黏膜上皮细胞,经光学和电子显微镜观察UU对其粘附性和致病性。结果：UU感染后,兔输卵管管腔有较多渗出物,呈淡红染匀质状,黏膜层的上皮细胞轻度肿胀,黏膜下层有炎症细胞(以淋巴细胞为主)呈灶性浸润;在电镜下,UU粘附于输卵管黏膜上皮细胞上,同时上皮细胞游离面的纤毛互相粘连、倒状,纤毛细胞核膜欠完整．胞核染色质呈凝块状．胞浆内可见大量大小不等的空泡,无纤毛细胞顶面参差不齐,可见伪足样突出．微绒毛减少．胞浆内近细胞顶部有少量分泌颗粒和空泡变性,细胞间可见相嵌连接。结论：UU到达兔输卵管后可粘附于输卵管黏膜上皮细胞上并导致损伤。     

Effects of species and cellular activity of oviductal epithelial cells on their dialogue with co-cultured mouse embryos

An efficient co-culture system, especially with oviductal or uterine epithelial cells, is important not only for the production of high quality embryos, but also for the study of the molecular dialogue between embryos and their maternal environment. Although mouse embryos have been co-cultured successfully with oviductal epithelial cells (OECs) from several species, studies on the effects of species and functionality of OECs are few. Reports concerning the necessity of direct contact between the embryo and OECs and about the culture of mouse embryos in medium conditioned with heterologous OECs have been controversial. In this study, pronuclear embryos from Kunming mice, characterized by an obvious two-cell block in vitro, were co-cultured with mouse, goat, and chick OECs. The functionality of OECs was determined by analyzing the cell cycle, apoptosis, the numbers of mitochondria and cilia, and the ability both to support embryonic development and to remove hypoxanthine from the culture medium. The necessity of direct contact between OECs and embryos was studied by repeated renewal of culture medium with fresh conditioned medium, the culture of embryos in plastic wells connected by tunnels to wells with OEC monolayers, and the co-culture of embryos separated from OECs by a filter. Both goat and chick OECs supported mouse embryonic development, but their embryotrophic lifespan was shorter than that of the mouse OECs. Whereas media conditioned with mouse OECs supported mouse embryonic development satisfactorily, medium conditioned with goat OECs supported little development. Immediate dialogue between heterologous OECs and embryos was essential for efficient co-culture, whereas direct contact between the two cell types was not; neither dialogue nor contact was needed between isologous OECs and embryos. Embryotrophic activity and the ability to remove hypoxanthine from conditioned medium declined with time after confluence and number of passages of OECs, mainly because of apoptosis and dedifferentiation. Thus, the species and functionality of OECs have profound effects on their molecular dialogue with co-cultured embryos, and efficient co-culture depends upon both positive and negative conditioning.This study was supported by grants from the China National Natural Science Foundation (nos. 30430530 and 30571337) and the “973” Project of the Chinese Science and Technology Ministry (no. G200016108).     

Extremely low frequency electromagnetic field exposure affects fertilization outcome in swine animal model

Modern society continuously exposes the population to electromagnetic radiation, the effects of which on human health, in particular reproduction, are still unknown. The aim of this research was to assess the effect of acute (1 h) exposure of boar spermatozoa to a 50 Hz extremely low frequency electromagnetic field (ELF-EMF) on early fertility outcome. The effect of intensities ranging from 0 to 2 mT on morpho-functional integrity of capacitated spermatozoa was examined in vitro. The oviducts containing or without spermatozoa were then exposed to the minimum in vivo, TD_50, and maximum intensities determined in vitro, 4 h before ovulation. The effects of ELF-EMF on spermatozoa in terms of early embryo development were evaluated after 12 h and 6 days. It was found that in vitro ELF-EMF >0.5 mT induced a progressive acrosome damage, thus compromising the ability of spermatozoa to undergo acrosomal reaction after zona pellucida stimulation and reducing the in vitro fertilization outcome. These effects became evident at 0.75 mT and reached the plateau at 1 mT. Under in vivo conditions, the ELF-EMF intensity of 1 mT was able to compromise sperm function, significantly reducing the fertilization rate. In addition, the exposure of oviducts to fields ≥ 0.75 mT in the absence of spermatozoa was able to negatively affect early embryo development. In fact, it was found to cause a slowdown in the embryo cleavage. In conclusion, it was demonstrated how and at which intensities ELF-EMF negatively affect early fertility outcome in a highly predictive animal model.