Rejuvenation of the disposable soma: Repeated injury extends lifespan in an asexual annelid

The disposable soma theory of senescence proposes that aging is the result of the accumulation of somatic damage with age resulting from insufficient somatic maintenance and repair. Comparative studies that show a positive correlation between longevity and DNA excision repair efficiency in mammals provide support for the theory but their validity has been questioned. A more satisfactory approach to investigate the role of somatic damage accumulation in aging would be to manipulate experimentally the levels of somatic repair and observe its effect on longevity. Here I report the results of studies in the asexual annelid Paranais litoralis where I have experimentally extended the worms' lifespan by subjecting them to repeated injury. I propose that repeated injury enhanced the normal level of repair of the worms, resulting in a rejuvenation of the soma. These results provide experimental support for the disposable soma theory of senescence.     

Telomere maintenance and telomerase activity are differentially regulated in asexual and sexual worms

In most sexually reproducing animals, replication and maintenance of telomeres occurs in the germ line and during early development in embryogenesis through the use of telomerase. Somatic cells generally do not maintain telomere sequences, and these cells become senescent in adults as telomeres shorten to a critical length. Some animals reproduce clonally and must therefore require adult somatic mechanisms for maintaining their chromosome ends. Here we study the telomere biology of planarian flatworms with apparently limitless regenerative capacity fueled by a population of highly proliferative adult stem cells. We show that somatic telomere maintenance is different in asexual and sexual animals. Asexual animals maintain telomere length somatically during reproduction by fission or when regeneration is induced by amputation, whereas sexual animals only achieve telomere elongation through sexual reproduction. We demonstrate that this difference is reflected in the expression and alternate splicing of the protein subunit of the telomerase enzyme. Asexual adult planarian stem cells appear to maintain telomere length over evolutionary timescales without passage through a germ-line stage. The adaptations we observe demonstrate indefinite somatic telomerase activity in proliferating stem cells during regeneration or reproduction by fission, and establish planarians as a pertinent model for studying telomere structure, function, and maintenance.     

Sex and ageing

Sex and ageing are often linked, particularly in the context of the evolutionary theories of ageing, which suggest that senescence may be the price for investing in offspring at the expense of somatic maintenance and repair. Considerable evidence supports this concept although, strictly, it is not sex per se but the existence of the soma/germ-line distinction that appears to hold the key. Other aspects of the sex-ageing axis seeing exciting new developments are the evolution of the human life history, particularly with respect to menopause, and the molecular mechanisms that sustain the immortality of the germ-line in contrast to the cumulative damage that appears to underlie the ageing of somatic cells.     

Reassessment of immortalization complementation group D

Previous somatic cell hybridization studies have assigned many human cell lines to one of four complementation groups (A-D) for immortalization. We report here that the A 1698DM cell line, which contains selectable markers and has previously been defined as the immortalization group D representative, was derived from T24 cells rather than A1698. A1698DM did not undergo senescence when fused with cell lines assigned to groups A, B, or C. This raises the possibility that this cell line has undergone further evolution and lost multiple putative senescence genes so that it is now unable to complement any, or most, other cell lines for senescence. Cell lines previously assigned to group D may, therefore, be heterogeneous with respect to the genetic changes that resulted in their immortalization. This has important implications for strategies to clone senescence genes based on complementation groups.     

Primary epimutations introduced during intracytoplasmic sperm injection (ICSI) are corrected by germline-specific epigenetic reprogramming

The use of assisted reproductive technologies (ART) has become increasingly common worldwide and is now responsible for 2-3% of children born in developed countries. Multiple reports have suggested that ART-conceived children are more likely to develop rare epigenetic disorders such as Beckwith-Wiedemann Syndrome or Angelman Syndrome, both of which involve dysregulation of imprinted genes. Anecdotal reports suggest that animals produced with ART that manifest apparent epigenetic defects typically do not transmit these epimutations to subsequent generations when allowed to breed naturally, but this hypothesis has not been directly studied. We analyzed allele-specific DNA methylation and expression at three imprinted genes, H19, Snrpn, and Peg3, in somatic cells from adult mice generated with the use of intracytoplasmic sperm injection (ICSI), a type of ART. Epimutations were detected in most of the ICSI-derived mice, but not in somatic cells of their offspring produced by natural mating. We examined germ cells from the ICSI mice that exhibited epimutations in their somatic cells and confirmed normal epigenetic reprogramming of the three imprinted genes analyzed. Collectively, these results confirm that ART procedures can lead to the formation of primary epimutations, but while such epimutations are likely to be maintained indefinitely in somatic cells of the ART-derived individuals, they are normally corrected in the germ line by epigenetic reprogramming and thus, not propagated to subsequent generations.     

nanos function is essential for development and regeneration of planarian germ cells

Germ cells are required for the successful propagation of sexually reproducing species. Understanding the mechanisms by which these cells are specified and how their totipotency is established and maintained has important biomedical and evolutionary implications. Freshwater planarians serve as fascinating models for studying these questions. They can regenerate germ cells from fragments of adult tissues that lack reproductive structures, suggesting that inductive signaling is involved in planarian germ cell specification. To study the development and regeneration of planarian germ cells, we have functionally characterized an ortholog of nanos, a gene required for germ cell development in diverse organisms, from Schmidtea mediterranea. In the hermaphroditic strain of this species, Smed-nanos mRNA is detected in developing, regenerating, and mature ovaries and testes. However, it is not detected in the vast majority of newly hatched planarians or in small tissue fragments that will ultimately regenerate germ cells, consistent with an epigenetic origin of germ cells. We show that Smed-nanos RNA interference (RNAi) results in failure to develop, regenerate, or maintain gonads in sexual planarians. Unexpectedly, Smed-nanos mRNA is also detected in presumptive testes primordia of asexual individuals that reproduce strictly by fission. These presumptive germ cells are lost after Smed-nanos RNAi, suggesting that asexual planarians specify germ cells, but their differentiation is blocked downstream of Smed-nanos function. Our results reveal a conserved function of nanos in germ cell development in planarians and suggest that these animals will serve as useful models for dissecting the molecular basis of epigenetic germ cell specification.     

Decrease in cytosine methylation at CpG island shores and increase in DNA fragmentation during zebrafish aging

Age-related changes in DNA methylation have been demonstrated in mammals, but it remains unclear as to the generality of this phenomenon in vertebrates, which is a criterion for the fundamental cause of senescence. Here we showed that the zebrafish genome gradually and clearly lost methylcytosine in somatic cells, but not in male germ cells during aging, and that age-dependent hypomethylation preferentially occurred at a particular domain called the CpG island shore, which is associated with vertebrates’ genes and has been shown to be hypomethylated in humans with age. We also found that two CpG island shores hypomethylated in zebrafish oocytes were de novo methylated in fertilized eggs, which suggests that the zebrafish epigenome is reset upon fertilization, enabling new generations to restart with a heavily methylated genome. Furthermore, we observed an increase in cleavage of the zebrafish genome to an oligonucleosome length in somatic cells from the age of 12 months, which is suggestive of an elevated rate of apoptosis in the senescent stage.     

Two-step oligoclonal development of male germ cells

During mouse development, primordial germ cells (PGCs) that give rise to the entire germ line are first identified within the proximal epiblast. However, long-term tracing of the fate of the cells has not been done wherein all cells in and around the germ-cell lineage are identified. Also, quantitative estimates of the number of founder PGCs using different models have come up with various numbers. Here, we use tetrachimeric mice to show that the progenitor numbers for the entire germ line in adult testis, and for the initiating embryonic PGCs, are both 4 cells. Although they proliferate to form polyclonal germ-cell populations in fetal and neonatal testes, germ cells that actually contribute to adult spermatogenesis originate from a small number of secondary founder cells that originate in the fetal period. The rest of the “deciduous” germ cells are lost, most likely by apoptosis, before the reproductive period. The second “actual” founder germ cells generally form small numbers of large monoclonal areas in testes by the reproductive period. Our results also demonstrate that there is no contribution of somatic cells to the male germ cell pool during development or in adulthood. These results suggest a model of 2-step oligoclonal development of male germ cells in mice, the second step distinguishing the heritable germ line from cells selected not to participate in forming the next generation.     

Germ and somatic cell lineages in the developing gonad

The germ cell lineage in the mouse becomes lineage-restricted about 7.2 days post coitum. Its progenitors have migrated from the proximal region of the epiblast, where they were subject to a predisposing signal from the adjacent extra-embryonic ectoderm. It appears that this and other signals determine the emergence of germ cells: unlike in some other organisms, this event is not pre-determined. After about 24 h in their initial extraembryonic location, the primordial germ cells migrate back into the embryo and make their way into the region of the developing gonad. Less is known about the origin of the various somatic cell lineages in the gonad, but some are known to derive from cells that migrate in from the mesonephros and others from the coelomic epithelium. Within the developing gonad, numerous interactions occur between the germ and somatic cell lineages. These are particularly important for the establishment of the spermatogenic lineage in the testis and for the differentiation of somatic tissue in the ovary. This paper will describe first the development of the germ cell lineage, up until about the time of birth, then the various somatic components of the gonad and finally the interactions that are known to occur between lineages. Unless otherwise stated, all the information refers to the mouse.     

Germ cell selection in genetic mosaics in Drosophila melanogaster

Heritable mutations in the germ line lead to genetically heterogeneous, or mosaic, gonads. Many of the genes used in germ-line development also play roles in somatic development [Saffman, E. E. & Lasko, P. (1999) Cell. Mol. Life Sci. 55, 1141-1163]. Mutations in these genes may have cellular phenotypes throughout germ-line development leading to their differential elimination or survival, as has been observed in somatic cells [Morata, G. & Ripoll, P. (1975) Dev. Biol. 42, 211-221]. We investigate whether mutations in heterozygosis are subject to pregametic selection in the germ line. We initiated clones of wild-type homozygous cells at different stages of development in gonads heterozygous for eight different recessive chromosome deficiencies. Here we show that cell selection takes place in mosaic germ-line populations. This phenomenon represents a level of selection that precedes and conditions subsequent zygotic selection by affecting the genes available in the gametic population.     

The mouse juvenile spermatogonial depletion (jsd) phenotype is due to a mutation in the X-derived retrogene, mUtp14b

The recessive juvenile spermatogonial depletion (jsd) mutation results in a single wave of spermatogenesis, followed by failure of type A spermatogonia to differentiate, resulting in adult male sterility. We have identified a jsd-specific rearrangement in the mouse homologue of the Saccharomyces cerevisiae gene UTP14, termed mUtp14b. Confirmation that mUtp14b underlies the jsd phenotype was obtained by transgenic bacterial artificial chromosome (BAC) rescue. We also identified a homologous gene on the Mus musculus X chromosome (MMUX) (mUtp14a) that is the strict homologue of the yeast gene, from which the intronless mUtp14b has been derived by retrotransposition. Expression analysis showed that mUtp14b is predominantly expressed in the germ line of the testis from zygotene through round spermatids, whereas mUtp14a, although well expressed in all somatic tissues, could be detected only in the germ line in round spermatids. In yeast, depletion of the UTP proteins impedes production of 18S rRNA, leading to cell death. We propose that the retroposed autosomal copy mUtp14b, having acquired a testis-specific expression pattern, could have provided a mechanism for increasing the efficiency and/or numbers of germ cells produced by meeting the need for more 18S rRNA and protein. Such a mechanism would be of obvious reproductive advantage and be strongly selected for in evolution. Consistent with this hypothesis is the finding of a similar X-autosome retroposition of UTP14 in human which seems to have arisen independently of that in rodents. In jsd homozygotes, which lack a functional copy of Utp14b, insufficient production of rRNA quickly leads to a cessation of spermatogenesis.     

Tumor suppressor gene Rb is required for self-renewal of spermatogonial stem cells in mice

The retinoblastoma tumor suppressor gene Rb is essential for maintaining the quiescence and for regulating the differentiation of somatic stem cells. Inactivation of Rb in somatic stem cells typically leads to their overexpansion, often followed by increased apoptosis, defective terminal differentiation, and tumor formation. However, Rb’s roles in germ-line stem cells have not been explored. We conditionally disrupted the Rb gene in mouse germ cells in vivo and discovered unanticipated consequences for GFRa1-protein-expressing A_single (GFRa1⁺ A_s) spermatogonia, the major source of male germ-line stem cells. Rb-deficient GFRa1⁺ A_s spermatogonia were present at normal density in testes 5 d after birth, but they lacked the capacity for self-renewal, resulting in germ cell depletion by 2 mo of age. Rb deficiency did not affect the proliferative activity of GFRa1⁺ A_s spermatogonia, but their progeny were exclusively transit-amplifying progenitor spermatogonia and did not include GFRa1⁺ A_s spermatogonia. In addition, Rb deficiency caused prolonged proliferation of progenitor spermatogonia, transiently enlarging this population. Despite these defects, Rb deficiency did not block terminal differentiation into functional sperm; offspring were readily obtained from young males whose germ cell pool was not yet depleted. We conclude that Rb is required for self-renewal of germ-line stem cells, but contrary to its critical roles in somatic stem cells, it is dispensable for their proliferative activity and terminal differentiation. Thus, this study identifies an unexpected function for Rb in maintaining the stem cell pool in the male germ line.     

X chromosome reactivation in oocytes of Mus caroli.

Mature mammalian oocytes have both of their X chromosomes active, while somatic cells from the same individual have one of their X chromosomes in an inactive state. We asked whether the X chromosomes of the germ cells never undergo inactivation in their ontogeny or whether inactivation of an X chromosome does occur but is followed by a subsequent reactivation event. Our approach has used an electrophoretic polymorphism for the X-linked enzyme glucose-6-phosphate dehydrogenase (G6PD) in the mouse species Mus caroli. G6PD is dimeric, and a heterodimer is produced in cells from heterozygous females if and only if both X chromosomes are active. Ovaries from heterozygous fetuses at different gestational ages were dissected and either studied cytologically or pressed between microscopy slides to obtain germ cell-rich and germ cell-poor preparations. No heterodimer band was detected on the 10th day of development in germ cell-rich preparations. On subsequent days, an increasingly intense heterodimer band was detected, which, by the 13th day, was approximately twice as intense as the corresponding homodimer bands. Consideration of (i) the G6PD activity per germ cell and per somatic cell and (ii) the percentage of germ cells in the germ cell-rich preparations indicated that a heterodimer band should have been visible on the 10th day had both X chromosomes been active. Cytological examinations showed that the earliest germ cells enter meiotic prophase on the eleventh day. These results demonstrate that oogonia have a single active X chromosome and that the inactive X chromosome is reactivated at or, more likely, shortly before entry into meiotic prophase.     

Irregular telomeres impair meiotic synapsis and recombination in mice

Telomere shortening can lead to chromosome instability, replicative senescence, and apoptosis in both somatic and male germ cells. To study roles for mammalian telomeres in homologous pairing and recombination, we characterized effects of telomere shortening on spermatogenesis and oogenesis in late-generation telomerase-deficient mice. We show that shortened telomeres of late-generation telomerase-deficient mice impair meiotic synapsis and decrease recombination, in particular, in females. In response to telomere shortening, male germ cells mostly undergo apoptosis, whereas female germ cells preferentially arrest in early meiosis, suggesting sexually dimorphic surveillance mechanisms for telomere dysfunction during meiosis in mice. Further, meiocytes of late-generation telomerase-deficient females with shortened telomeres, bred with early-generation males harboring relatively long telomeres, exhibit severely impaired chromosome pairing and synapsis and reduced meiotic recombination. These findings imply that functional telomeres are important in mammalian meiotic synapsis and recombination.