Large Scale Identification of Proteins, Mucins, and Their O-Glycosylation in the Endocervical Mucus during the Menstrual Cycle

Abstract

The mucus filling the human cervical opening blocks the entry to the uterus, but this has to be relative and allow for the sperm to penetrate at ovulation. We studied this mucus, its content of proteins and mucins, and the mucin O-glycosylation in cervical secretions before, during, and after ovulation. Cervical mucosal secretions from 12 subjects were collected, reduced-alkylated, separated with polyacrylamide or agarose/polyacrylamide gel electrophoresis, and stained with silver, Alcian blue, or Coomassie Blue stain. Protein and mucin bands from before and during ovulation were digested and subsequently analyzed by nano-LC-FT-ICR MS and MS/MS. We identified 194 proteins after searches against the NCBI non-redundant protein database and an in-house mucin database. Three gel-forming (MUC5B, MUC5AC, and MUC6) and two transmembrane mucins (MUC16 and MUC1) were identified. For the analysis of mucin O-glycosylation, separated mucins from six individuals were blotted to PVDF membranes, and the O-glycans were released by reductive ␤-elimination and analyzed with capillary HPLC-MS and -MS/MS. At least 50 neutral, sialic acid-, and sulfate-containing oligosaccharides were found. An increase of GlcNAc-6GalNAcol Core 2 structures and a relative decrease of NeuAc residues are typical for ovulation, and NeuAc6GalNAcol and NeuAc-3Gal-epitopes are typical for the non-ovulatory phases. The cervical mucus at ovulation is thus characterized by a relative increase in neutral fucosylated oligosaccharides. This comprehensive characterization of the mucus during the menstrual cycle suggests mucin glycosylation as the major alteration at ovulation, but the relation to the altered physicochemical properties and sperm penetrability is still not understood.

Culturing and Applications of Rotating Wall Vessel Bioreactor Derived 3D Epithelial Cell Models

“…Vaginal 3-D aggregates expressed a lower percentage of MUC1 (27.7%) compared to monolayers (67.2%). The lower percentage of MUC1 on the 3-D aggregates surface compared to monolayers may be a result of the majority of MUC1 being secreted from the cells as observed in the vaginal tract 17,18 .…”

“…We and others have shown that RWV-derived models utilizing human cells may be a valuable tool for predicting the efficacy, toxicity and pharmacokinetics of vaccines, microbicides, biologics and pharmaceuticals in a fashion that is relevant to humans 1,2,18 . With the success of this bioreactor culture system to produce authentic human responses, combined with its flexibility, the applications of the RWV bioreactor system is currently being expanded to such fields as tumor modeling, regenerative medicine, and tissue engineering 2,[18][19][20][21][22][23] .…”

“…microvilli), and cellular polarity. The majority of the data and examples presented here are using vaginal epithelial cells grown in the RWV system, however the RWV system also has been used to culture other epithelial cell types including small and large intestine, lung, bladder, liver, and tonsil epithelial cells to form 3-D organotypic models 1,[7][8][9][10][11][12][13]18 . Additionally, this system has been used to culture cell types other than epithelial cells, and currently the development of complex, multicellular organotypic culture models are underway 2,18 .…”

“…With the success of this bioreactor culture system to produce authentic human responses, combined with its flexibility, the applications of the RWV bioreactor system is currently being expanded to such fields as tumor modeling, regenerative medicine, and tissue engineering 2,[18][19][20][21][22][23] . To advance our RWV-generated mucosal models we are working to create a more complex multicellular system that replicates both the structure and function of the human mucosal tissue.…”

Conflicting Views on the Molecular Structure of the Cancer Antigen CA125/MUC16

“…Thus, the challenge of discovering MUC16/CA125 structure necessitates integrated and cooperative efforts. Recent studies have been undertaken by investigators who have focused on analysis of this biomarker from different biological sources by proteomic approaches using the MALDI-TOF mass spectrometry [70,72], the tandem mass spectrometry [MS/MS] [68,73] or the nano-LC-FT-ICR MS [74,75]. Although mucins are difficult to identify with proteomics as most of their peptides are glycosylated and thus not identified in the protein databases [72], implementation of such approaches is likely to yield more information about the complexity of MUC16/CA125 and to achieve a substantial depth of analysis.…”