Journal: PLoS ONE

Article Title: The Testicular and Epididymal Expression Profile of PLCζ in Mouse and Human Does Not Support Its Role as a Sperm-Borne Oocyte Activating Factor

doi: 10.1371/journal.pone.0033496

Figure Lengend Snippet: ( A ) Detergent extraction of mouse cauda epididymal spermatozoa. The ∼74 kDa band corresponding to the functional isoform of PLCζ (arrow) was detected in whole sperm (WS) and was resistant to NP40 extraction. Anti-hmPLCζ was used to detect the signal with a similar result obtained with anti-EF. ( B ) The NP40 resistant sperm fraction ( i.e. , pellet in panel A) was sonicated and sperm heads and tails were separated through a sucrose gradient. The 74 kDa active isoform was found only in the tail fraction (arrow). Further incubation of the tail fraction with Triton x-100/DTT (Tail Tx-DTT) extracted all of the PLCζ bands, suggesting the mitochondrial sheath of the mid piece as the origin of PLCζ in the sperm tail. Anti-EF was used to detect the signal with a similar result obtained with anti- hmPLCζ. Lanes were run on the same gel but were non-contiguous. ( C ) Human sperm extraction by NP40. The functional isoform (arrow) was detergent extractable as shown in NP40 lane. Anti-hmPLCζ was used for detection. A similar result was observed with anti-EF and anti-hPLCζ. WS, Whole sperm; Sc.Sn, Sonication supernatant.

Article Snippet: Additionally for human spermatozoa, we used the polyclonal rabbit antiserum against two peptides of human PLCζ (C- RESKSYFNPSNIKE -coNH 2 ; C- ETHERKGSDKRGDN -coNH 2 ), initially obtained from Dr. John Parrington, University of Oxford, and later purchased from Covalab (Villeurbanne, France), named as anti-hPLCζ.

Techniques: Extraction, Functional Assay, Sonication, Incubation

Journal: PLoS ONE

Article Title: The Testicular and Epididymal Expression Profile of PLCζ in Mouse and Human Does Not Support Its Role as a Sperm-Borne Oocyte Activating Factor

doi: 10.1371/journal.pone.0033496

Figure Lengend Snippet: ( A ) PLCζ (red) localized to the forming acrosome in round spermatid (top) and elongated spermatid (bottom) of testicular spreads. DNA was stained by DAPI (blue). ( B, C ) Mature spermatozoa were extracted from cauda epididymis and ejaculate in mouse and human, respectively. The PLCζ immunofluorescence was detected in both non-permeabilized and permeabilized mouse (B) and human (C) spermatozoa. NP40 extraction abolished the detected signal on the sperm head, confirming the presence of PLCζ on the surface of mature spermatozoa. Immunolocalization above was done with anti-EF with a similar result obtained with anti-hmPLCζ. DNA staining is with DAPI in A (blue) and SYTOX green in B (green), DIC; differential interference contrast. Bars = 5 µm.

Article Snippet: Additionally for human spermatozoa, we used the polyclonal rabbit antiserum against two peptides of human PLCζ (C- RESKSYFNPSNIKE -coNH 2 ; C- ETHERKGSDKRGDN -coNH 2 ), initially obtained from Dr. John Parrington, University of Oxford, and later purchased from Covalab (Villeurbanne, France), named as anti-hPLCζ.

Techniques: Staining, Immunofluorescence, Extraction

Journal: PLoS ONE

Article Title: The Testicular and Epididymal Expression Profile of PLCζ in Mouse and Human Does Not Support Its Role as a Sperm-Borne Oocyte Activating Factor

doi: 10.1371/journal.pone.0033496

Figure Lengend Snippet: Note the PLCζ immunoreactivity (red) in the acrosome region during sperm-zona binding. The immunoreactivity disappears along with acrosome shroud (arrows) when the spermatozoa undergo the acrosome reaction. No PLCζ immunoreactivity was detectable on the sperm heads during sperm-oolemma binding and incorporation into the oocyte. The results shown here are from anti-pPLCζ while a similar result was observed with anti-EF. DAPI (blue) was used for DNA staining; DIC, Differential Interference Contrast. Bars = 25 µm.

Article Snippet: Additionally for human spermatozoa, we used the polyclonal rabbit antiserum against two peptides of human PLCζ (C- RESKSYFNPSNIKE -coNH 2 ; C- ETHERKGSDKRGDN -coNH 2 ), initially obtained from Dr. John Parrington, University of Oxford, and later purchased from Covalab (Villeurbanne, France), named as anti-hPLCζ.

Techniques: Binding Assay, Staining

Journal:

Article Title: Aberrant expression of homeobox gene HOXA7 is associated with m?llerian-like differentiation of epithelial ovarian tumors and the generation of a specific autologous antibody response

doi: 10.1073/pnas.011503998

Figure Lengend Snippet: Serum antibody responses to HOXA7. (A) Antibodies to HOXA7 in sera (diluted 1:500) of patients with ovarian carcinomas (ca.) (blots 1–9) and with ovarian cystadenomas (blots 10, 11), and of healthy female volunteers (blots 12–14), were detected by their reactivity to plaques of monoclonalized HOXA7 positive phage. Patient code numbers are shown with the type and degree of histology of their tumors. Histology of carcinomas included those of the serous and endometrioid subtypes, which were poorly, moderately (mod.), and well differentiated (diff.). Background reactivity to plaques of monoclonalized HOXA7 positive phage of the secondary anti-human IgG antibody is shown in blot 15. (B) Reactivity of sera (1:500 dilution) with recombinant HOXA7 protein (dark bars) and to the negative control protein BPVL2 (light bars) was assessed by ELISA and measured in terms of optical density at 450 nm. Shown are values of statistical significance, as determined by the Mann–Whitney u test, for differences in reactivity to HOXA7 of sera of patients with moderately differentiated serous ovarian carcinomas (n = 24), with poorly differentiated serous ovarian carcinomas (n = 24), with serous ovarian cystadenomas (n = 19), and of healthy female volunteers (n = 30). Differences in reactivity to BPVL2 among the four groups of women were found to be not significant (P > 0.05). Horizontal bars indicate median values.

Article Snippet: Immunohistochemical analysis was performed on sections of paraffin-embedded tissue specimens by using a rabbit polyclonal antiserum raised against human HOXA7 peptide (Covance) (1:2,000) in a standard avidin–biotin–peroxidase method.

Techniques: Recombinant, Negative Control, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal:

Article Title: Aberrant expression of homeobox gene HOXA7 is associated with m?llerian-like differentiation of epithelial ovarian tumors and the generation of a specific autologous antibody response

doi: 10.1073/pnas.011503998

Figure Lengend Snippet: Expression patterns of HOXA7 in benign and malignant epithelial ovarian tumors. (A) Shown are Southern blots of HOXA7 and β-actin RT-PCR products amplified from RNA of ovarian carcinomas (lanes 1–15, 16, 21), in ovarian cystadenomas (lanes 17–20), in epithelial cells scraped off surfaces of normal ovaries (lanes 22–24), and in the simian virus 40-immortalized OSE cell line, IOSE-29 (lane 25). The specimen used for analysis shown in lane 4 (case SCM4) is the same as those in lanes 16 and 21. Specimens included those collected from the same patients whose sera were tested for reactivity to HOXA7 by the phage plaque assay shown in Fig. ​Fig.11A (refer to corresponding patient code numbers). (B) Shown are typical examples of intense staining using HOXA7 antibody in a histologically differentiated serous carcinoma (case SCM4) and serous cystadenoma (case SCB2), and of weak staining in poorly differentiated serous (case SCP2) and endometrioid (case ECP1) carcinomas. (×60.)

Article Snippet: Immunohistochemical analysis was performed on sections of paraffin-embedded tissue specimens by using a rabbit polyclonal antiserum raised against human HOXA7 peptide (Covance) (1:2,000) in a standard avidin–biotin–peroxidase method.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Amplification, Plaque Assay, Staining

Journal:

Article Title: Aberrant expression of homeobox gene HOXA7 is associated with m?llerian-like differentiation of epithelial ovarian tumors and the generation of a specific autologous antibody response

doi: 10.1073/pnas.011503998

Figure Lengend Snippet: Analysis of HOXA7 expression in normal tissue. Shown are examples of minimal HOXA7 staining in cells lining the ovarian surface (A), increased staining in columnar cells lining invaginations of the ovarian surface (B), and intense staining in the epithelium lining inclusion cysts of histologically normal ovaries (C and D). Strong HOXA7 staining was observed throughout the epithelium of normal fallopian tubes (E), and was similar in intensity and uniformity to the staining observed in serous cystadenomas (F). (A–C, ×400; D–F, ×160.)

Article Snippet: Immunohistochemical analysis was performed on sections of paraffin-embedded tissue specimens by using a rabbit polyclonal antiserum raised against human HOXA7 peptide (Covance) (1:2,000) in a standard avidin–biotin–peroxidase method.

Techniques: Expressing, Staining

Journal:

Article Title: Aberrant expression of homeobox gene HOXA7 is associated with m?llerian-like differentiation of epithelial ovarian tumors and the generation of a specific autologous antibody response

doi: 10.1073/pnas.011503998

Figure Lengend Snippet: Ectopic HOXA7 expression in immortalized OSE cells. Cultures of parental IOSE-29 cells (A, E, I), IOSE-29 cells transfected with pcDNA4His vector (B, F, J), IOSE-29 cells transfected with pcDNA4His-HOXA7 construct (C, G, K), and OVCAR-3 cells (D, H, L) were photographed by phase-contrast microscopy (A–D), or fixed and stained by immunofluorescence for E-cadherin (E–H) and for vimentin (I–L). (A–D, ×140; E–L, ×640.)

Article Snippet: Immunohistochemical analysis was performed on sections of paraffin-embedded tissue specimens by using a rabbit polyclonal antiserum raised against human HOXA7 peptide (Covance) (1:2,000) in a standard avidin–biotin–peroxidase method.

Techniques: Expressing, Transfection, Plasmid Preparation, Construct, Microscopy, Staining, Immunofluorescence