New monoclonal antibodies specific for mammalian protamines P1 and P2

ABSTRACT

The expression of protamines and the binding of these small arginine-rich proteins to DNA complete the process of spermatid chromatin reorganization and the global inactivation of the male’s haploid genome that occurs during the final stages of sperm development in mammals. While a number of anti-protamine antibodies have been created during the last 40 years, only a few have proven useful for detecting the presence of the protamines, determining the timing of their expression and deposition in chromatin, and investigating their structure and function in both maturing spermatids and sperm. The aim of this effort was to develop an additional set of monoclonal antibodies (MAbs) that not only recognize new P1 and P2 protamine epitopes but also work well as IHC reagents for detecting and identifying mammalian protamines in testicular tissue and ejaculated sperm. Using a combination of native and synthetic human protamines as antigens, 38 hybridoma clones recognizing human protamine P1 or P2 were generated. Antibodies produced by the 12 best clones were screened for selectivity by enzyme-linked immunosorbent assay, and two were found to recognize only human protamine P1 or P2, while a number of the others bound to both the human and mouse proteins. One MAb recognized every protamine tested. All the antibodies, including one recognizing stallion P1 and another recognizing stallion P2, bound to the native protamines in the chromatin of spermatids or sperm. While the majority labeled only elongating spermatids or sperm, several of the antibodies were found to also bind to the cytoplasm or nuclei of cells that lack protamine, which indicates these MAbs must recognize epitopes present in the protamines that are also found in other proteins. Thirteen overlapping human protamine P1 peptides were synthesized and subsequently used to identify the epitopes recognized by the six best antibodies.

Abbreviations: BSA: bovine serum albumin; ELISA: enzyme-linked immunosorbent assay; HCl: hydrochloric acid; IHC: immunohistochemistry; i.p: intraperitoneal; LIS: lithium diiodosalicylate; MAb: monoclonal antibody; PBS: phosphate buffered saline

KEYWORDS:

• Protamine P1
• protamine P2
• anti-protamine monoclonal antibodies
• mammalian protamine antibodies
• stallion protamine antibodies

Acknowledgments

We are grateful to Barbara Fröhlich and Tania Bloch for performing the immunohistochemical labeling of the testicular tissue and semen samples, respectively. This work was supported in part by a grant from the University Clinic Giessen and Marburg (UKGM) 29/2015 GI to KS and HCS and funding provided by Briar Patch Biosciences LLC to RB and MCB. The authors would also like to acknowledge Dr. Gordon Dixon for his many important and insightful contributions to the field of sperm chromatin research. His research, which spanned more than 50 years, provided the foundation for what we now know about the structure of sperm chromatin and the role the protamines and other nuclear proteins play in its function.

Disclosure statement

R. Balhorn and M. C. Balhorn are founders of Briar Patch Biosciences, LLC, the company that provided the funding for the development of the antibodies characterized in this study. K. Steger, M. Bergmann, H.-C. Schuppe and S. Neuhauser report no declarations of interest.

Supplementary data

Supplemental data for this article can be accessed here.

Additional information

Funding

This work was supported by the University Clinic Giessen and Marburg [29/2015 GI] and Briar Patch Biosciences LLC [U9140BF130].

Notes on contributors

Rod Balhorn

Isolated or prepared antigens, developed strategy for antigen immunizations and antibody development, analyzed data, wrote the paper: RB; Directed and analyzed IHC labeling of human, mouse, and stallion testicular tissues and human and stallion semen, contributed to writing the paper: KS; Tissue fixation and embedding, histological evaluation of human tissue samples: MB; Obtained ethics committee approval for the use of the tissues included in this study and performed semen analyses according to the WHO recommendations: HCS; Collected stallion semen samples: SN; Analyzed data, contributed to the writing and editing of the paper, and prepared the final forms of the figures: MCB.