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.     

Proteome analysis of chick embryonic cerebrospinal fluid

During early stages of embryo development, the brain cavity is filled with embryonic cerebrospinal fluid (E-CSF), a complex fluid containing different protein fractions that contributes to the regulation of the survival, proliferation and neurogenesis of the neuroectodermal stem cells. Using 2-DE, protein sequencing and database searches, we identified and analyzed the proteome of the E-CSF from chick embryos (Gallus gallus). We identified 26 different gene products, including proteins related to the extracellular matrix, proteins associated with the regulation of osmotic pressure and metal transport, proteins related to cell survival, MAP kinase activators, proteins involved in the transport of retinol and vitamin D, antioxidant and antimicrobial proteins, intracellular proteins and some unknown proteins. Most of these gene products are involved in the regulation of developmental processes during embryogenesis in systems other than E-CSF. Interestingly, 14 of them are also present in adult human CSF proteome, and it has been reported that they are altered in the CSF of patients suffering neurodegenerative diseases and/or neurological disorders. Understanding these molecules and the mechanisms they control during embryonic neurogenesis is a key contribution to the general understanding of CNS development, and may also contribute to greater knowledge of these human diseases.     

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.     

Proteome analysis of human metaphase chromosomes

DNA is packaged as chromatin in the interphase nucleus. During mitosis, chromatin fibers are highly condensed to form metaphase chromosomes, which ensure equal segregation of replicated chromosomal DNA into the daughter cells. Despite >1 century of research on metaphase chromosomes, information regarding the higher order structure of metaphase chromosomes is limited, and it is still not clear which proteins are involved in further folding of the chromatin fiber into metaphase chromosomes. To obtain a global view of the chromosomal proteins, we performed proteome analyses on three types of isolated human metaphase chromosomes. We first show the results from comparative proteome analyses of two types of isolated human metaphase chromosomes that have been frequently used in biochemical and morphological analyses. 209 proteins were quantitatively identified and classified into six groups on the basis of their known interphase localization. Furthermore, a list of 107 proteins was obtained from the proteome analyses of highly purified metaphase chromosomes, the majority of which are essential for chromosome structure and function. Based on the information obtained on these proteins and on their localizations during mitosis as assessed by immunostaining, we present a four-layer model of metaphase chromosomes. According to this model, the chromosomal proteins have been newly classified into each of four groups: chromosome coating proteins, chromosome peripheral proteins, chromosome structural proteins, and chromosome fibrous proteins. This analysis represents the first compositional view of human metaphase chromosomes and provides a protein framework for future research on this topic.     

Proteome analysis of resting human neutrophils

Neutrophils constitute the first line of host defense against pathogens. In the present study 2-D gel electrophoresis-mass spectrometry technology was employed to analyze the human resting neutrophils proteome. One hundred and two conserved spots were subjected to peptide mass fingerprinting, yielding 22 identifications. Among the identified proteins, nine are related to the inflammatory process, two polypeptides are assigned to metabolic functions and five are classified as structural.     

Source of prolactin in human follicular fluid.

To analyze whether prolactin (PRL) in human follicular fluid (FF) is synthesized locally or derived from the circulation, PRL concentrations of plasma and FF were determined in the patients after ovarian stimulations. The amounts of PRL messenger ribonucleic acid (mRNA) in the follicular tissues during different menstrual phases were also determined. The FF PRL concentration was correlated positively with plasma PRL and highest estradiol levels during the stimulatory cycle. No PRL mRNA sequence was detected in the RNAs extracted from follicles at any stage in the menstrual cycle, although beta-actin mRNA was detected in all samples. In a comparison with pituitary RNA, the PRL mRNA concentration in ovarian follicular tissues seemed to be 10,000 times less than that in the pituitary. These results suggest that FF PRL may not be synthesized locally, but derived from the pituitary via the circulation through passive diffusion, and thus regulated by estrogen.     

Expression of proopiomelanocortin-related peptides in human follicular fluid

In order to evaluate the expression of the opioid precursor proopiomelanocortin (POMC) in the ovarian follicle, we measured 6 of its main end-products in 23 follicular fluids. We coupled high performance liquid chromatography (HPLC) to specific radioimmunoassays. Seven follicles were immature (diameter less than 9 mm), 10 were obtained from superovulated patients during an in vitro fertilization-embryo transfer program (greater than 22 mm) and six were persistent follicles, collected during the luteal phase [15-31 mm, luteinized unruptured follicles (LUF)]. Follicular fluids were extracted by mean of Sep-pak cartridges and then purified by HPLC with a reverse-phase C-18 column eluted in a linear gradient with acetonitrile/0.01 M hydrochloric acid (from 18:82 to 40:60). Fractions were tested with specific antisera for ACTH (1-39), alpha-MSH, beta-lipotropin (beta-LPH), beta-endorphin (beta-EP) and gamma-endorphin (gamma-EP) immunoreactivities. No presence of beta-LPH, beta-EP and ACTH was confirmed, while gamma-EP, alpha-MSH and des-alpha-MSH were detected for the first time in follicular fluid. In every class of follicles shorter chain peptides predominate over their longer chain precursor. Immature follicles are characterized by the highest amounts of gamma-EP, ACTH, alpha-MSH and des-alpha-MSH if compared to superovulated and LUF. On the contrary, beta-EP amount was highest after superovulation. Apart from this finding, peptide levels in superovulated patients and LUF are similar.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proteome analysis of human lung squamous carcinoma

Few lung cancer-specific molecular markers have been established in regard of "early-stage" diagnosis and prognosis. In this study the proteome analysis of human lung squamous carcinoma (hLSC) was carried out using two strategies to explore the carcinogenic mechanisms and identify its molecular markers more directly and comprehensively. Comparative proteome analysis on 20 hLSC tissues and paired normal bronchial epithelial tissues revealed 76 differential proteins, among which 68 proteins were identified by PMF. The identified proteins fell into three categories: oncoproteins, cell cycle regulators and signaling molecules. To validate the identified differential proteins, the expressions levels of three differential proteins mdm2, c-jun and EGFR were determined by immunohistochemical staining and immunoblots. The results verified proteome analysis results. Serological proteome analysis (SERPA) of ten hLSC tissues was performed to identify the tumor-associated antigens. The results revealed 36 +/- 8 differential proteins reactive with patients' autologous sera, of which 14 proteins were identified. Six of the 14 proteins, alpha enolase, pre-B cell-enhancing factor precursor, triosephosphate isomerase, phosphoglycerate mutase 1, fructose-bisphosphate aldolase A, and guanine nucleotide-binding protein beta subunit-like protein, were also up-regulated in hLSCs in the comparative proteomic study, which suggests potential application of these 6 hLSC-associated antigens in diagnosis and therapy of hLSC.     

Proteome analysis of the human mitotic spindle

The accurate distribution of sister chromatids during cell division is crucial for the generation of two cells with the same complement of genetic information. A highly dynamic microtubule-based structure, the mitotic spindle, carries out the physical separation of the chromosomes to opposite poles of the cells and, moreover, determines the cell division cleavage plane. In animal cells, the spindle comprises microtubules that radiate from the microtubule organizing centers, the centrosomes, and interact with kinetochores on the chromosomes. Malfunctioning of the spindle can lead to chromosome missegregation and hence result in aneuploidy, a hallmark of most human cancers. Despite major progress in deciphering the temporal and spatial regulation of the mitotic spindle, its composition and function are not fully understood. A more complete inventory of spindle components would therefore constitute an important advance. Here we describe the purification of human mitotic spindles and their analysis by MS/MS. We identified 151 proteins previously known to associate with the spindle apparatus, centrosomes, and/or kinetochores and 644 other proteins, including 154 uncharacterized components that did not show obvious homologies to known proteins and did not contain motifs indicative of a particular localization. Of these uncharacterized proteins, 17 were tagged and localized in transfected mitotic cells, resulting in the identification of six genuine spindle components (KIAA0008, CdcA8, KIAA1187, FLJ12649, FLJ90806, and C20Orf129). This study illustrates the strength of a proteomic approach for the analysis of isolated human spindles and identifies several novel spindle components for future functional studies.     

Proteome analysis of haptoglobin in cerebrospinal fluid of neuromyelitis optica

Neuromyelitis optica (NMO) is an inflammatory demyelinating disease with generally poor prognosis that selectively targets optic nerves and spinal cord. Although diagnostic criteria for NMO are available, there is still a need for biomarkers, predicting disease development and progression to improve individually tailored treatment. CSF proteins were separated by two-dimensional electrophoresis and identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). The interaction between these proteins was further analyzed by Pathway Studio software. Seven protein spots in CSF were significantly altered in NMO patients compared with controls. Identification made by mass spectrometry revealed that the most significant protein was haptoglobin, which was increased in the NMO gels. The subsequent ELISA test were performed to validate it, which confirmed the results of proteomic analysis. Protein network was built, which showed some biological interactions among the seven proteins. These results support a correlation between the level of haptoglobin and NMO. Haptoglobin may be a potential useful biomarker for diagnosis or a medicine target for treatment of NMO.     

Proteome analysis of X-ray irradiated human erythroleukemia cells

In order to discover molecular biomarkers in radiation response we investigated the effects of X-radiation on radioresistant K562 cells by using a comparative proteomic analysis. In treated cells 29 up-regulated and 10 down-regulated proteins were detected by image analysis and identified by mass spectrometry. Elongation factor 1 alpha 1 and stress-70 protein showed a 6.2 and 5.4 fold increase respectively in treated cells. Additional proteins such us pi and omega classes glutathione transferases, ATP synthase D chain, were also found to be up-regulated, suggesting that the enzyme belonging to the cellular detoxification system against oxidative stress and energetic metabolism may have a key role in the cellular response to radiation injury. This data set may provide a useful tool to design a combined chemo- and radiotherapic strategy against leukemia disease.     

Proteome analysis of pitcher fluid of the carnivorous plant Nepenthes alata

The genus Nepenthes comprises carnivorous plants that digest insects in pitcher fluid to supplement their nitrogen uptake. In a recent study, two acid proteinases (nepenthesins I and II) were purified from the pitcher fluid. However, no other enzymes involved in prey digestion have been identified, although several enzyme activities have been reported. To identify all the proteins involved, we performed a proteomic analysis of Nepenthes pitcher fluid. The secreted proteins in pitcher fluid were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and several protein bands were detected by silver staining. The proteins were identified by in-gel tryptic digestion, de novo peptide sequencing, and homology searches against public databases. The proteins included homologues of beta-D-xylosidase, beta-1,3-glucanase, chitinase, and thaumatin-like protein, most of which are designated "pathogenesis-related proteins". These proteins presumably inhibit bacterial growth in the pitcher fluid to ensure sufficient nutrients for Nepenthes growth.     

Haptoglobin inhibits lecithin-cholesterol acyltransferase in human ovarian follicular fluid

The activity of the enzyme lecithin-cholesterol acyltransferase (LCAT; E.C. 2.3.1.43) is involved in the removal of cholesterol excess from peripheral cells. This activity is stimulated by the HDL (high density lipoprotein) apolipoprotein A1 (ApoA1). Haptoglobin (Hpt) was previously found to be associated with ApoA1 in ovarian follicular fluid. LCAT activity was analyzed in follicular fluids, collected from an IVF program, containing different amounts of Hpt or Hpt/ApoA1 ratio. Addition of purified Hpt to follicular fluid caused a decrease in the enzyme activity, which was measured as the rate of synthesis of cholesteryl esters. In the fractions of fluid proteins, as obtained by gel filtration chromatography, Hpt and HDL were titrated by ELISA while the LCAT activity was assayed by using radioactive cholesterol and purified HDL. When isolated LCAT was incubated with fractions containing different Hpt/ApoA1 ratios, the enzyme activity was found negatively correlated with the Hpt/ApoA1 ratio (P < 0.01). LCAT kinetic parameters were measured in two fractions with the same amount of ApoA1 (5 microg/ml) but different amounts of Hpt (0.69 or 3.77 microg/ml): the V(max) did not change while the K(m) values were 24.1 or 78.6 microM in the presence of the low or high Hpt level, respectively. The analysis of fluids associated with cytoplasmically mature MII oocytes, in a cross-sectional study, confirmed that a negative correlation exists between the Hpt/ApoA1 ratio and the LCAT activity (P < 0.01). The results suggest that Hpt inhibits the reverse transport of cholesterol by preventing ApoA1 stimulation of the LCAT activity.     

Proteome analysis of human substantia nigra in Parkinson's disease

Protein expression has been compared in human substantia nigra specimens from Parkinson's disease (PD) patients and from controls, and 44 proteins expressed in this midbrain region were identified by peptide mass fingerprinting. Among them, nine showed changes in their abundance. L and M neurofilament chains are less abundant in PD specimens, whereas peroxiredoxin II, mitochondrial complex III, ATP synthase D chain, complexin I, profilin, L-type calcium channel delta-subunit, and fatty-acid binding protein are significantly more present in PD samples than in controls. Besides the consolidated view of oxidative stress involvement in PD pathogenesis, suggested by overexpression of mitochondrial and reactive oxygen species (ROS)-scavenging proteins, these results indicate a possible potentiation mechanism of afferent signals to substantia nigra following degeneration of dopaminergic neurons.     

Immunoglobulins from human follicular fluid induce the acrosome reaction in human sperm

The ability of human follicular fluid (hFF), retrieved from women undergoing IVF to induce the acrosome reaction (AR) in human sperm has been documented by several laboratories. However, the nature of the active factors in the hFF and the physiological meaning of the AR induction are highly controversial. We performed a three step purification scheme for hFF and all the fractions were screened for the AR-inducing activity. AR activity was associated with a protein fraction of M_r > 180 kD that on further analysis under PAGE was found to be composed by subunits of apparent M_r 50,000 and 29,000. The N-terminal sequences of these bands showed a 100% homology with the heavy and light chains of human lgG. A polyclonal antibody raised against the purified protein and anti-human lgG were both able to suppress the acrosome reactioninducing activity of crude hFF. However, neither normal human serum nor a purified preparation of human lgG were able to mimic the AR-inducing activity of hFF. We concluded that the AR-inducing activity of hFF is, at least in part, due to the presence of antisperm antibodies. © 1994 Wiley-Liss, Inc.     

Proteome analysis of human substantia nigra in Parkinson's disease

Background  

Parkinson's disease (PD) is the most common neurodegenerative disorder involving the motor system. Although not being the only region involved in PD, affection of the substantia nigra and its projections is responsible for some of the most debilitating features of the disease. To further advance a comprehensive understanding of nigral pathology, we conducted a tissue based comparative proteome study of healthy and diseased human substantia nigra.     

Endocrine parameters of human follicular fluid and fertilization capacity of oocytes

The induction of multiple follicular growth during ovarian stimulation for in vitro fertilization (IVF) implies follicular asynchrony. As a consequence oocytes of different quality are obtained. The maturity and fertilizability of oocytes cannot sufficiently be predicted by their morphological appearance under the light microscope. Looking for additional parameters of oocyte quality, FSH, hCG, estradiol (E2), progesterone (P), testosterone (T), prolactin (PRL) and cAMP were analysed in human follicular fluid (FF) containing a morphologically mature oocyte. The evaluation of the relationship between FF values and oocyte fertilization showed the following results: no differences of FSH, hCG, E2, P and T concentrations in FF between the group of fertilized and not fertilized ova. However, significant differences were detected for PRL and cAMP. In FF of fertilized oocytes PRL content was higher (38.8 +/- 2.2 vs 29.7 +/- 2.3 ng/ml, P less than 0.01) and cAMP level was lower (32.7 +/- 1.9 vs 59.8 +/- 7.4 pmol/ml, P less than 0.01) as compared with FF of unfertilizable oocytes. In conclusion PRL- and cAMP concentration of FF might be additional parameters of oocyte maturation and fertilizability.