BRCA1 and BRCA2 protein expressions in an ovotestis of a 46, XX true hermaphrodite

Abstract

breast cancer susceptibility genes encode proteins, the normal cellular functions of which are complex and multiple, and germ-line mutations in individuals predispose both to breast and to ovarian cancer. There is nevertheless substantial evidence linking BRCA1 and BRCA2 to homologous recombination and DNA repair, to transcriptional control and to tissue proliferation. There is controversy regarding the localization of BRCA1 and BRCA2 proteins to either nucleus or cytoplasm and whether the expression is present in premeiotic germ cells or can still be expressed in mitotic spermatogonia. We report herein an immunohistochemical study of BRCA1 and BRCA2 distribution in a rather unsual tissue (an ovotestis), which addresses this issue.

Introduction

], and a moderate increased risk for the development of ovarian cancer.

transcripts were localized specifically to granulosa cells, thecal cells and oocytes of developing follicles as well as luteal cells of recently formed corpora lutea and surface epithelium.

Considering these results, we further investigated the presence of human BRCA1 and BRCA2 proteins in an ovotestis by immunochemical analysis with a different panel of antibodies against BRCA1 and BRCA2.

Materials and methods

). This paper presents our laboratory findings concerning the BRCA1 and BRCA2 protein expression in this particular gonad.

.

Results and discussion

].

-transferase (GST)-BRCA1 fusion protein containing amino acids encoded by a 3´ portion of BRCA1 exon 11 and by a 5´ portion of BRCA1 exon 11, respectively. These antibodies seemed to remain more specific, and they exhibited nuclear staining.

].

The antibody for BRCA2 (66066E) recognizes epitopes between amino acids 1323-1346 of human BRCA2. The antibody 66076E recognizes epitopes between amino acids 2586-2600. Antibodies were purchased from PharMingen (San Diego, CA, USA) and tested by Western blotting in HBL-100 human breast cells to ensure they recognized the 390 kDa BRCA2 protein. Both BRCA2 antibodies also cross-reacted with smaller proteins, which could be degradation products.

]. Using the 3E6 antibodies, we detected a protein of 390 kDa, in MCF-7 human tumor breast cell lysates, which corresponds to the predicted size of the 3418 amino acid BRCA2 sequence. We also detected, in CCL 221 colorectal adenocarcinoma cell lysates, a single band at 390 kDa using the 5F6 antibodies (data not shown).

) was exhibited in male and female germ cells, and low nuclear staining was found in Sertoli cells.

). With 5F6 antibodies (data not shown), exclusively cytoplasmic staining was obtained for BRCA2 protein in the male compartment, and low intensive nuclear and cytoplasmic stainings were obtained in oocytes and follicles.

The differences of staining patterns for the same protein in the same tissue may be explained by the choice of the antibodies. The monoclonal antibodies raised against GST-BRCA1 or GST-BRCA2 fusion proteins seem more specific than antibodies raised against a 20 amino acid peptide.

Conclusion

genes.

Acknowledgement

The authors are grateful to Christelle Picard and Jacqueline Avinain for technical assistance, and Guy Ragonnaud for prints.

Sponsorship

This research was supported by La Ligue Nationale Française de Lutte Contre le Cancer and Le Comité du Puy-de-Dôme. CV is a recipient of MENRT (Ministère de l'Education Nationale, de la Recherche et de la Technologie) funding.

Figures and Tables

3E6 antibodies showed low cytoplasmic staining of Sertoli cells (asterisk) and female germ cells (arrowhead) (× 660).

Specificity of the primary antibodies used, and corresponding subcellular localization and degree of staining observed for ovotestis

mouse monoclonal antibodies. aa, Amino acid; Cyt., cytoplasm; N, nuclear; (+) intensive; (++), very intensive.

Keywords

• BRCA1
• BRCA2
• immunohistochemistry
• ovotestis