With few exceptions, the Arf
tumor suppressor is not expressed in normal tissues of healthy mice but is induced by abnormally sustained and elevated thresholds of proliferative signals, activating a p53 response that opposes the deleterious effects of oncogene activation. Notably, p53 responds to a much wider range of Arf
-independent signal transduction cascades triggered by many other forms of cellular stress, including acute DNA damage, to which the Arf
promoter does not respond 
. By converging on p53, these different signaling pathways inhibit cell cycle progression or trigger apoptosis, acting to suppress tumor formation.
We now document a physiological role of Arf in mouse male germ cell development that is distinct from its tumor suppressive functions in key respects. First, Arf is expressed in spermatogonia, but not in the primary spermatocytes that arise from them. Expression of p19Arf neither arrests spermatogonial mitotic progression nor triggers their p53-dependent apoptosis. However, the absence of Arf expression in spermatogonia leads to p53-dependent apoptosis of spermatocytes before they exit meiosis-I. The defect in spermatogenesis is germ cell autonomous and results in a significant reduction in sperm counts by the time Arf-null mice are two months old, although residual sperm production maintains fertility in young males. Thus, expression of Arf in mitotic progenitor cells enhances the survival of their meiotic progeny in which Arf expression is normally extinguished. These features indicate that Arf expression initiates a salutary, feed-forward program that facilitates meiotic progression. Indeed, although Arf and Ink4a are widely viewed to convey tumor suppressive functions that coordinate the activities of the p53 and Rb signaling “pathways,” inactivation of Arf and Ink4a in the testes leads to opposing outcomes. Disruption of Ink4a increases the mitotic activity of spermatogonial progenitors to enhance sperm output and, in this respect, compensates for Arf loss of function without eliminating the cellular defects that arise in the Arf-null setting. In short, loss of Ink4a increases the spermatogonial pool size, but without Arf expression, spermatocytes undergo increased apoptosis, returning the number of mature sperm to normal levels.
Homologous recombination during meiosis exchanges genetic information between maternally and paternally derived chromosomes and also guides proper segregation of chromosome pairs to maintain correct chromosome numbers in gametes 
. During meiosis, in contrast to mitotically diving cells, homologous chromosomes are favored over sister chromatids as templates for recombinational DNA repair. Double-strand DNA breaks are formed by the topoisomerase-II-related transesterase Spo11. This process activates the Atm kinase and leads to phosphorylation of the H2AX histone variant near sites of strand breakage during early prophase I. Binding of the RecA family strand exchange proteins, Rad51 and meiosis-specific Dmc1, to Spo11-induced DNA ends generates filaments that search for and invade homologous duplex DNA molecules, leading to pairing of homologous chromosomes. Loading of Rad51 and Dmc1 is normally reversed by early pachytene when chromosomes are fully synapsed, after which γ-H2AX foci are no longer detected.
In the Arf-null setting, a modest but significant increase in γ-H2AX staining was first detected in the least mature spermatogonia, and primary spermatocytes displayed accentuated signals that persisted inappropriately into the pachytene stage. Arf-null cells also formed fewer Dmc1/Rad51 foci at zygotene and exhibited focal regions of asynapsis at pachytene. Aberrant Arf-null spermatocytes underwent apoptosis at pachytene, resulting in the emergence of fewer diplotene cells and a significant reduction in sperm output. Importantly, Arf−/−; p53−/− double-null pachytene cells also exhibited persistent γ-H2AX staining, but these cells escaped elimination. Thus, apoptosis was p53-dependent, but aberrant γ-H2AX accumulation was not.
Although the underlying mechanisms remain unknown, we consider here two plausible interpretations of this apoptotic arrest. First, it may be that reduced Rad51/Dmc1 focus formation and persistent γ-H2AX staining in Arf
-null male germ cells connote a defect in DNA repair that then activates p53 through Arf
-independent but Atm/Atr-dependent signaling pathways. In this scenario, Spo11-induced DSBs would form at normal levels but Rad51/Dmc1 loading would be impaired such that some DNA damage would persist into pachytene. This might conceivably involve the p53-independent ability of p19Arf
to promote the sumoylation of numerous target proteins by inhibiting the SUMO2/3 protease Senp3 
. SUMO2/3 accumulates at sites of DNA damage in mammalian cells 
, and various aspects of DNA repair are regulated by the SUMO conjugation pathway 
. There is fragmentary evidence that absence of p19Arf
compromises nucleotide excision repair in cultured cells 
raising the possibility that Arf
may play an as yet undefined role in promoting homologous recombination. All meiotic mutants that cannot properly synapse homologous chromosomes arrest during pachytene 
, and accompanying defects in sex body formation and failure to properly silence transcription of the sex chromosomes during prophase is itself sufficient to eliminate pachytene cells 
. However, spermatocytes can also undergo apoptosis in direct response to unrepaired Spo11-induced breaks even if sex body formation is normal 
. Where tested, spermatocyte apoptosis in meiotic mutants with chromosome synapsis errors has been found to be p53-independent 
. Moreover, Spo11-dependent activated phospho-p53 can be transiently detected from leptonema and zygonema in wild-type male mice, and in Drosophila
, p53 activity is prolonged in cells defective for meiotic repair 
. Thus, it remains a formal possibility that meiotic recombination defects can trigger p53-dependent apoptosis.
A second, alternative interpretation rests on the idea that the earlier and less profound accumulation of γ-H2AX in Arf
-null spermatogonia might be a symptom of an underlying defect affecting chromatin structure or Atm/Atr signaling. The appearance of γ-H2AX reflects chromatin modifications that flank sites of DNA damage rather than strand breaks themselves, so the kinetics of γ-H2AX formation and dissolution do not necessarily coincide with the appearance and repair of DNA damage 
. Moreover, aberrant Atm/Atr signaling is itself sufficient to activate p53, whether triggered by DNA breaks or not 
. Thus, it may be that Arf
deficiency causes inappropriate Atm/Atr signaling that provokes p53-dependent apoptosis in a DNA damage-independent manner. In this view, the observed meiotic prophase defects in Arf
-null spermatocytes may possibly be a separate downstream consequence of this earlier anomaly, and may not be the cause of apoptosis. Regardless of which interpretation is correct, it is important to note that our findings provide strong evidence that p53-dependent monitoring promotes proper meiotic maturation, in addition to the previously documented p53-independent pathway(s). Whatever the underlying mechanisms, the role of Arf
in male germ cell development contrasts with the general paradigm of p19Arf
acting as an activator of p53. Instead, it is the absence of Arf
in spermatogonia that consequently leads to p53-dependent apoptosis of spermatocytes.