During vegetative growth, budding yeast reproduces by asymmetric division giving rise to a daughter cell, which first emerges from the mother as an outgrowth, known as a bud. The number of daughter cells a mother cell generates before its death is known as its replicative life span and is relatively uniform for a given strain (Sinclair et al., 1998
). With the ongoing of replicative life, mitotic yeast cells show morphological and physiological changes such as an increase in cell size, genome instability, sterility, extension in cell cycle duration, nucleolar fragmentation, accumulation of ROS (reactive oxygen species), and loss of membrane turgescence. Several factors contribute to these changes in the cell physiology associated with senescence: damaged proteins, damaged mitochondria, redistribution of the silencing regulator proteins Sir2, Sir3 and Sir4 and the accumulation of ERCs (extrachromosomal ribosomal DNA circles). ERCs are formed through homologous recombination and are self-replicating, but they lack centromeres and therefore remain in the mother cell during cell division. Interestingly, damaged proteins or damaged mitochondria are also preferentially retained in the mother cell during mitosis. These observations have led to the suggestion that it is the accumulation of ERCs and damaged proteins and organelles that lead to cell death in aged cells (Sinclair and Mills, 2001).
Several genes have been implicated in regulating replicative life span in yeast. The histone deacetylase Sir2 modulates aging in virtually every organism. In budding yeast, deletion of SIR2
severely shortens life span, whereas the presence of an additional copy of this gene increases the lifespan by approximately 50% (Kaeberlein et al., 1999
is thought to affect aging by preventing ERC formation through its role in repressing mitotic recombination within the rDNA locus. The rDNA localized Fob1 protein also controls life span in yeast. Fob1 prevents movement of DNA polymerases against the direction of rRNA transcription thereby reducing recombination within the rDNA (Defossez et al., 1999
Nutrient signaling also regulates life span. The TOR (target of rapamycin) kinases are highly conserved and promote cell growth in response to favorable nutrient conditions and growth-factor signals. Budding yeast contains two TOR kinases, Tor1 and Tor2. TOR2
is essential, but deletion of TOR1
has been shown to increase replicative life span by approximately 20% (Kaeberlein et al., 2005
). It has been proposed that it is through TOR1
that caloric restriction delays aging and leads to an increase in life span in budding yeast. The protein kinase Sch9 is phosphorylated by Tor1 and thought to convey some of Tor1's growth promoting functions (Urban et al., 2007
). Consistent with a role of caloric restriction and the TOR pathway in the regulation of replicative life span, cells lacking SCH9
live longer (Kaeberlein et al., 2005
The effects of age on entry into and progression through meiosis are largely unexplored. In humans, meiotic chromosome segregation errors increase with maternal age (reviewed in Hassold and Hunt, 2001
). Approximately 80% of these segregation errors occur during meiosis I, and 20% result from meiosis II non-disjunction (Sherman et al., 2005
). Studies on chromosome 21 non-disjunction show that only 6–10% of all trisomy 21 cases are due to errors in spermatogenesis, but meiosis I and meiosis II errors contribute equally to these male germline non-disjunction events (Sherman et al, 2005
). Additionally, there is also evidence to suggest that sperm quality decreases with age (Malaspina et al., 2001
; Wyrobek et al., 2006
). A gradual increase in DNA damage or a reduced ability to protect germ line stem cells from free radicals has been suggested to be the basis for this decrease in sperm quality (Zhu et al., 2007
). However, how replicative age affects the meiotic divisions has not been studied in detail in any organism. The ability to isolate aged yeast cells (Smeal et al, 1996
) and to induce them to undergo meiosis (Honigberg and Purnapatre, 2003
) enabled us to address this question.
In budding yeast four different signals are necessary for cells to enter the meiotic program. Cells must express both mating type loci, and nitrogen and glucose must be absent from the growth medium. Finally, cells must be respiration competent (Honigberg and Purnapatre, 2003
). The mating-type, nutritional, and respiration signals converge at Ime1, a transcription factor governing entry into the meiotic program. This regulation occurs at multiple levels and is still largely unknown. The MATa and α genes are needed together to inactivate the transcriptional repressor Rme1 (Covitz et al., 1991
), which acts at the IME1
promoter (Sagee et al., 1998
). The fermentable carbon source glucose inhibits IME1
transcription the mechanisms of which is only partly understood (Gorner et al., 1998
; Shenhar et al., 2001
). Nitrogen also prevents IME1
transcription (Sagee et al., 1998
), prevents Ime1 from localizing to the nucleus (Colomina et al., 2003
) and disrupts its interaction with its coactivator Ume6 (Xiao and Mitchell, 2000
). The respiration state of a cell also affects IME1
expression. Cells lacking functional mitochondria fail to express IME1
(Treinin et al., 1993
; Jambhekar and Amon, 2008
), but the mechanisms whereby this occurs remain to be determined.
Once cells have entered the meiotic program, they undergo pre-meiotic DNA replication, which is followed by two rounds of chromosome segregation. During the ensuing prophase, linkages are created between homologous chromosomes through recombination. This facilitates the correct alignment and the subsequent segregation of homologous chromosomes during the first meiotic division (meiosis I). This unusual segregation phase is immediately followed by a second division, meiosis II, which resembles mitosis in that sister chromatids are segregated. After completion of meiosis II, all four meiotic products are packaged into spores.
In this study, we adapted previously established protocols to isolate aged mother cells to examine the effects of age on the ability of cells to enter and progress through meiosis. We find that aged mother cells fail to enter the meiotic program. This inability to initiate meiosis is accompanied by a failure to induce IME1 and other early meiotic genes. Ectopic expression of IME1 partially suppresses the sporulation defect indicating that age inhibits meiosis in part by preventing IME1 expression. We then describe the identification of conditions that partially alleviate the meiotic entry defect in aged cells, which enabled us to examine the consequences of cellular age on subsequent meiotic stages. When the meiotic entry defect is suppressed, aged mother cells complete the meiotic program but exhibit defects in meiotic chromosome segregation and spore formation. Finally, we show that mutations that extend the replicative life of budding yeast suppress the meiotic defects of aged cells. Our results indicate that in budding yeast, cellular age affects multiple aspects of the meiotic program and that mutations that extend the ability of cells to reproduce asexually also extend the ability to reproduce sexually.