Bertrand Evrard

Expression of Antimicrobial Defensins in the Male Reproductive Tract of Rats, Mice, and Humans

Abstract Defensins are antimicrobial peptides that play a major role in innate immunity. Using reverse transcriptase-polymerase chain reaction, immunochemistry, or both, we performed a search of all presently known defensins in rat testis, epididymis, and isolated testicular cells; in mouse testis and epididymis; and in human testis and ejaculates. In the rat, all α- and β-defensins except RNP-4 were expressed within the testis, whereas α-defensins RNP1–2, RNP-4, and β-defensins RBD-1 and RBD-2 were present within the epididymis. In the mouse, the cryptdin transcripts CRS1C, mBD-1, and mBD-2 were detected in the testis and epididymis, whereas mBD-3 and mBD-4 were expressed only in the epididymis, and CRS4C was absent in both organs. In the human testis, transcripts for four known defensins were expressed with the consistent exception of HBD-2 and HBD-3. In rat interstitial tissue, resident macrophages expressed most of the defensins studied, whereas Leydig cells produced only RBD-2. In contrast, all studied defensins except RNP-4 were present in the seminiferous tubules. Within these tubules, peritubular and Sertoli cells expressed most of the studied α- and β-defensins, whereas spermatogonia displayed only α-defensins, but at relatively high levels. Meiotic pachytene spermatocytes expressed only β-defensins, whereas postmeiotic spermatids and their cytoplasmic lobes displayed both types. In humans, the HBD-1 peptide was expressed mainly in the germ line from pachytene spermatocytes to late spermatids. The peptide was also present in ejaculated spermatozoa and seminal plasma, where multiple soluble forms were present. Finally, high salt concentration or dithiothreitol-sensitive cationic extracts from human seminal plasma were indeed found to display antimicrobial activity. We conclude that the male reproductive tract produces defensins that most probably assume an important, innate organ defense system against pathogens.

New Insights into the Rat Spermatogonial Proteome Identification of 156 Additional Proteins

Despite the essential role played by spermatogonia in testicular function, little is known about these cells. To improve our understanding of their biology, our group recently identified a set of 53 spermatogonial proteins using two-dimensional (2-D) gel electrophoresis and mass spectrometry. To continue this work, we investigated a subset of the spermatogonial proteome using narrow range immobilized pH gradients to favor the detection of less abundant proteins. A 2-D reference map of spermatogonia in the pH range 4–9 was created, and protein entities fractionated in a pH 5–6 2-D gel were further processed for protein identification. A new set of 156 polypeptides was identified by peptide mass fingerprinting and tandem mass spectrometry. These polypeptides corresponded to 102 different proteins, which reflect the complexity of post-translational modifications. Seventy-nine of these proteins were identified for the first time in spermatogonia. All identified proteins were classified into functional groups. This work represents a first step toward the establishment of a systematic spermatogonia protein database.

Testis hormone-sensitive lipase expression in spermatids is governed by a short promoter in transgenic mice.

A testicular form of hormone-sensitive lipase (HSLtes), a triacylglycerol lipase, and cholesterol esterase, is expressed in male germ cells. Northern blot analysis showed HSLtes mRNA expression in early spermatids. Immunolocalization of the protein in human and rodent seminiferous tubules indicated that the highest level of expression occurred in elongated spermatids. We have previously shown that 0.5 kilobase pairs of the human HSLtes promoter directs testis-specific expression of a chloramphenicol acetyltransferase reporter gene in transgenic mice and determined regions binding nuclear proteins expressed in testis but not in liver (Blaise, R., Grober, J., Rouet, P., Tavernier, G., Daegelen, D., and Langin, D. (1999)J. Biol. Chem. 274, 9327–9334). Mutation of a SRY/Sox-binding site in one of the regions did not impair in vivo testis-specific expression of the reporter gene. Further transgenic analyses established that 95 base pairs upstream of the transcription start site were sufficient for correct testis expression. In gel retardation assays using early spermatid nuclear extracts, a germ cell-specific DNA-protein interaction was mapped between −46 and −29 base pairs. The DNA binding nuclear protein showed properties of zinc finger transcription factors. Mutation of the region abolished reporter gene activity in transgenic mice, showing that it is necessary for testis expression of HSLtes.

Direct Iterative Protein Profiling (DIPP) - an Innovative Method for Large-scale Protein Detection Applied to Budding Yeast Mitosis

The budding yeast Saccharomyces cerevisiae is a major model organism for important biological processes such as mitotic growth and meiotic development, it can be a human pathogen, and it is widely used in the food-, and biotechnology industries. Consequently, the genomes of numerous strains have been sequenced and a very large amount of RNA profiling data is available. Moreover, it has recently become possible to quantitatively analyze the entire yeast proteome; however, efficient and cost-effective high-throughput protein profiling remains a challenge. We report here a new approach to direct and label-free large-scale yeast protein identification using a tandem buffer system for protein extraction, two-step protein prefractionation and enzymatic digestion, and detection of peptides by iterative mass spectrometry. Our profiling study of diploid cells undergoing rapid mitotic growth identified 86% of the known proteins and its output was found to be widely concordant with genome-wide mRNA concentrations and DNA variations between yeast strains. This paves the way for comprehensive and straightforward yeast proteome profiling across a wide variety of experimental conditions.