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First published online 27 May 2008
doi: 10.1242/jcs.030437

Journal of Cell Science 121, 2037-2045 (2008)
Published by The Company of Biologists 2008
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Nuclear pore complex proteins mark the implantation window in human endometrium

Elisa Guffanti1, Nupur Kittur1, Z. Nilly Brodt1, Alex J. Polotsky2, Satu M. Kuokkanen2, Debra S. Heller3, Steven L. Young4, Nanette Santoro2 and U. Thomas Meier1,*

1 Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA
2 Department of Obstetrics, Gynecology and Women's Health, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA
3 Department of Pathology, UMDNJ – New Jersey Medical School, Newark, NJ 07101, USA
4 Department of Obstetrics and Gynecology, University of North Carolina School of Medicine, Chapel Hill, NC 27599, USA


Figure 1
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  		Fig. 1. The monoclonal antibody 414 (mAb414) directed against nuclear pore complex (NPC) proteins exhibits a strong preference for NCSs. (A) Double fluorescence of mAb414 (A) and DAPI DNA stain (A") on a semi-thin frozen section of human endometrium in the secretory phase. NCS fluorescence appears as rings (A, arrows). The rings, i.e. the matrix and membrane tubules of NCSs, appear as phase-dense circles in phase contrast microscopy (A', arrows). Moreover, NCSs are often encircled by nucleoli (arrowheads) and, like nucleoli, appear chromatin-free (A"). The concentration of mAb414 antigens in NCSs is so high that the classical rim staining of NPCs only becomes visible if the image is overexposed to an extent that saturates NCS staining (A'"). (B) MAb414 immunogold-stained electron micrograph of an ultrathin cryosection of luteal human endometrium. Note the strong and specific gold labeling of a grazing section of a NCS (i.e. its core is covered by its membrane tubules and matrix) that is embedded in a nucleolus (No) and attached to the nuclear envelope (NE). At least one NPC of a neighboring cell is identified by mAb414 (arrow). (C) Confocal micrograph of indirect mAb414 fluorescence of a 7-µm-thick paraffin section of luteal human endometrium. In a single 0.2 µm optical section, a NCS is visible in only one of the nuclei defined by the classical rim staining of NPCs (C), whereas, in a maximum projection of all optical planes, all nuclei outlined by hazy NPC staining contain NCSs (C'). Scale bars: 5 µm.

 

Figure 2
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  		Fig. 2. NCSs are specific to human endometrium. Indirect mAb414 fluorescence on paraffin sections of various human (A-J) and baboon tissues (K,L). (A-J) Samples from commercial tissue arrays. One example out of six specimens each of human breast (A), lung (B), kidney (C), liver (D), esophagus (E), stomach (F), colon (G), and rectum (H) tissue. One example out of 59 specimens each of human control endometrium (I) and endometria with adenocarcinomas (J). (K,L) Two examples out of 19 baboon secretory endometrial biopsies. Note only control human secretory endometrium contains NCSs (arrows). Nuclear pore complex staining is generally better in endometria but, despite high background labeling, can be distinguished in the other tissues and should therefore reveal NCSs if present. Scale bar: 10 µm.

 

Figure 3
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  		Fig. 3. NCSs consist of a unique subset of NPC, and nuclear membrane and lamina proteins. Indirect immunofluorescence on semi-thin frozen sections of human luteal endometrium of antigens clearly present and/or enriched in NCSs (left column: A,C,E,G,I,K,M), of antigens absent from, barely detectable, or only in some NCSs (middle column: B,D,F,H,J,L,N), and of antigens clearly present in NCSs as double fluorescence control (right column: B',D',F',H',J',L',N'). The identity of all antigens is indicated on each panel. NCSs that are not obvious (E) and all in the double fluorescence series (two right columns) are indicated (arrows). In all cases, the identity of NCSs was confirmed by double fluorescence and/or phase-contrast microscopy. Note that although mAb414 recognizes all four nucleoporins, only Nup153 (A) and Nup62 (C) but not Nup358 (B) nor Nup214 (D) are present in NCSs. Tpr is present in only some (F, arrow) but not other NCSs (arrowheads). Of the two inner nuclear membrane and lamina-associated proteins emerin (G) and LAP2β (J), only emerin is enriched in NCSs. Nucleoli, identified by fibrillarin (N, arrowheads), are often adjacent to or surrounding NCSs (N', arrows) but do not overlap. Note the particularly high enrichment in NCSs of Nup153 (A), emerin (G) and lamin A/C (I), which at this exposure are barely detectable in their usual nuclear envelope locations. Magnification is identical in all panels; scale bar, 5 µm.

 

Figure 4
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  		Fig. 4. The NCS marks the implantation window. (A) Histogram of 64 human endometrial biopsies collected on the indicated luteal days (LH+) and scored for the percentage of epithelial cell nuclei containing NCSs using three categories, none (0%), less than 10% (<10%), and between 10% and 60% but mostly around 50% (~50%). Where available, the luteal day was determined in the following order of priority, according to LH surge, classical histological criteria (+) (Noyes et al., 1950Go), and chronological day (*). Some biopsies from conditions that may affect dating are indicated: (a) fibroid uterus; (b) menopause transition treated with hyper estrogen and hypo progesterone; (c) 30-34 day cycle; (d) 34-37 day cycle; (e) dysmenorrhea. The biopsies in which NCSs were quantified more accurately (Table 1) are marked (black dots). (B) Representative mAb414 fluorescence micrographs for each category in (A) including a proliferative biopsy. Scale bar: 20 µm. (C) Summary of the data in A expressed as a percentage of biopsies on each luteal day containing NCSs (black squares, left y-axis) and the number of biopsies analyzed on each day (gray circles, right y-axis). Note that only on luteal days 4-10 did over 70% of biopsies contain NCSs.

 

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© The Company of Biologists Ltd 2008