Although we often tend to attribute infertility to dysfunction of the female reproductive process, approximately 50% of cases are likely to result from abnormalities in spermatogenesis, or male sex cell production. After puberty, spermatogenesis continues throughout the life of a male mammal, proceeding from mitotic division of spermatogonia, through meiotic division of spermatocytes, to the transformation of haploid spermatids into mature, motile sperm (spermiogenesis). Of course, the integrity of the genome must be maintained throughout the process of spermatogenesis, as any sperm cell has the potential to contribute its DNA to the next generation. Levels of proteins involved in detection of DNA damage and in DNA repair are very high in the germ cell precursors for sperm and eggs, and remain elevated in spermatocytes up until a transition period that precedes spermiogenesis. After meiosis is completed, haploid spermatids enter a period during which they are repair-deficient, and the condensed DNA is transcriptionally inactive, and inaccessible to DNA repair enzymes. Thus, it is critical for any DNA damage, incurred during crossing over and other phases of meiosis, to be detected and repaired prior to spermiogenesis.
Sequence of events in mammalian spermatogenesis, with timeline for human and mouse. Day 0 = day of fertilization. From Marchetti and Wyrobek (2005)
Spermatogenesis in mice is very similar to that in humans, and the availability of mouse strains with targeted mutations in genes required for DNA damage detection and repair has facilitated analyses of genomic integrity maintenance during this process. In the 2007 paper I have chosen for my PLoS ONE@Two post, Paul and colleagues (http://www.plosone.org/article/info%3Adoi&2F10.1371%2Fjournal.pone.0000989) used three different strains of knockout mice, each of which harbored a targeted mutation in a different DNA repair gene, to examine the consequences of these deficiencies on male germ cell development:
1) The mismatch repair (MMR) pathway removes errors made during DNA replication that have escaped proofreading by DNA polymerase, and the MutS homologue 2 gene (Msh2) encodes a protein that is expressed at high levels in mouse spermatogonia and spermatocytes. Msh2-/- mice are more likely to develop skin cancer when exposed to UVB light, but have no known defects in spermatogenesis.
2) The nucleotide excision repair (NER) pathway takes care of UV-induced DNA damage, as well as bulky DNA lesions. The protein encoded by the Ercc1 gene (excision repair cross-complementing protein) is an essential component of NER, and is expressed at high levels in pachytene spermatocytes and round spermatids. Mice that lack this gene die of liver failure before spermatogenesis, but a transgenic “trick” allowed rescue of the liver phenotype. The rescued mice (TG-Ercc1) still lack ERCC1 in the testes, and are infertile.
3) Like ERCC1, the protein encoded by the p53 tumor suppressor gene is also involved in the homologous repair (HR) pathway. The p53 protein is expressed at high levels in the testes, and is important in recognition and repair of DNA strand breaks, as well as in mediating DNA damage-induced apoptosis. Mice that lack p53 are tumor-prone, and their mature sperm are less motile.
Double strand breaks in pachytene spermatocytes. Immunodetection of SCP3 (red) for synaptonemal complexes and γH2AX (green) for DNA DSB. The sex bodies are indicated by white arrowheads. From Paul et al. (2007)
To compare spermatogenesis and DNA damage in mice deficient for either MSH2, ERCC1, or p53, Paul and colleagues used a variety of techniques. First, they simply looked at the architecture of the seminiferous tubules in the testes, using standard histological staining methods. The TG-Ercc1-/- mice had reduced numbers of germ cell precursors, as did the Msh2-/- mice. Reduced diameters of seminiferous tubules were also observed in the testes of both these strains of knockout mice. To determine whether programmed cell death, or apoptosis, was elevated in the testes of the knockout mice, Paul and colleagues stained testicular sections for cleaved caspase-3. Increased numbers of apoptotic spermatogonia were observed in the TG-Ercc1-/- and p53-/- mice.
DNA damage detection in epididymal sperm, using the SCSA. From Paul et al. (2007)
Antibodies against the histone variant γH2AX can be used to detect unsynapsed DNA, as well as DNA double strand breaks, during meiosis. In normal pachytene spermatocytes, this variant is expected to bind to the sex body, which contains unpaired regions of the X and Y chromosomes. However, elevated numbers of γH2AX foci were detected in spermatocytes from TG-Ercc1-/-, p53-/-, and p53+/- mice. Moreover, another assay for DNA strand breaks and chromosome abnormalities, the Sperm Chromatin Structure Assay (SCSA), revealed increased DNA damage in epididymal sperm from TG-Ercc1-/- mice. Clearly, ERCC1 is critical for normal spermatogenesis, though results from other investigators indicate that the HR, rather than the NER, role of the protein is more important in this context. The functions of p53 in spermatogenesis are less clear, and confounded by the strain differences in infertility for p53-/- male mice. In the present study, on the CBA background, both DNA damage and apoptosis appeared to be elevated. The Msh2-/- mice exhibited early decreases in the number of germ cell precursors, but insignificant reductions in epididymal sperm numbers later on. Studies like this one, on the roles of DNA damage detection and repair pathway components during defined periods of spermatogenesis, are crucial to understanding how genomic integrity in the male germ line affects fertility and pregnancy outcomes. Moreover, such experiments provide a baseline for comparing the effects of environmental mutagens on reproduction and embryonic development.
Marchetti, F and Wyrobek, AJ (2005) Mechanisms and consequences of paternally-transmitted chromosomal abnormalities. Birth Defects Res. (Part C) 75, 112-129
Catriona Paul, Joanne E. Povey, Nicola J. Lawrence, Jim Selfridge, David W. Melton, Philippa T. K. Saunders (2007). Deletion of Genes Implicated in Protecting the Integrity of Male Germ Cells Has Differential Effects on the Incidence of DNA Breaks and Germ Cell Loss PLoS ONE, 2 (10) DOI: 10.1371/journal.pone.0000989