Introduction
This research line is funded by a project of the National R&D Plan (Ministry of Science and Innovation), led by Dr. Felipe Martínez-Pastor.
Sperm work has improved animal breeding and allowed semen banks to preserving species and breeds. However, many factors affect the integrity of the genetic material of the spermatozoon, reducing fertility, causing abortions and affecting offspring fitness. In this research line, we are studying ruminant spermatozoa, a group of great economical importance. Whereas artificial reproductive techniques are routine in cattle, they are still developing for most species. In either cases, it is crucial to maintain sperm DNA integrity during manipulation, storage or in vitro techniques. Sperm DNA assessment has been carried out for more than 30 years, but few studies have dealt on fine analysis of DNA damage. DNA in mammal sperm is associated to protamines (PDNA) and histones (HDNA), an organization with likely epigenetic effects. HDNA include important sequences (telomeres and genes involved in early development), and may be more vulnerable to insults, asking for dedicated analyses.
Likewise, apoptotic pathways may ultimately cause DNA damage. Caspase activation has been studied in spermatozoa from several species, but recently poly (ADP-ribose) polymerase (PARP), which has been detected in human semen, has been proposed as a new target. It may be linked to failed spermatogenesis or activated apoptosis pathways, and could be a harbinger of DNA damage.
Krawetz SA et al. 2009. Molecular aspects of male fertility. International Workshop on Molecular Andrology. EMBO Rep 10:1087-92.
Work plan
As a first step in this project, we are setting up several molecular biology techniques for studying ruminant spermatozoa (ram and red deer), in order to:
- carry out an specific analysis of several DNA sequences and telomeres to detect damage while discriminating HDNA and PDNA sequences.
- detect the cleaved form of PARP (cPARP), to use it as an apoptosis and DNA-damage marker.
Most studies have dealt with human sperm, thus we are testing the association of DNA sequences to histones and the presence of PARP in ruminant spermatozoa.
Then, we are studying the impact of stressing and protective treatments, to test if they affect the spermatozoa concerning DNA damage and PARP cleavage. During these experiments, we will test the association among PARP presence and cleavage with DNA damage assessed by qPCR, determining if PARP could have a role on the induction of such damage. Finally, we will analyse samples from a germplasm bank with fertility records (ram samples), assessing the impact of DNA damage on fertility, and the value of PARP evaluation as a fertility marker.
Expected outcome
The contributions of this project could be highly valuable. Not only it could greatly improve our understanding of the development of DNA damage and apoptosis-like processes in spermatozoa from non-human species, but it might also bring about practical consequences for ruminant breeding. The use of PARP as a new sperm quality marker could improve diagnostic and prognostic tests on semen. Moreover, studying DNA damage by qPCR and discriminating among regions with more or less vulnerability might help improving sperm work and the results of in vitro reproductive techniques.