Academics

Introduction

Telomeres are made up of a repeating hexanucleotide (TTAGGG in mammals) sequence that has been found to have an important role in protecting DNA during its replication cycle.  Specifically, telomeres prevent DNA terminal fusions and degradation (Tanemura et al., 2005).  When DNA polymerase synthesizes DNA, it does so in chunks going from the 5' to 3' end of the chromosome, but cannot replicate the very end of the molecule where the telomeres are.  Thus the telomerase ribonucleoprotein complex is needed to maintain telomere length (Coussens et al., 2006).  Telomerase is made up of a nine nucleotide RNA sequence (the length can vary depending on the species) that serves as a template for telomere repair (Blackburn et al., 2006) and the telomerase reverse transcriptase (TERT) enzyme (Coussens et al., 2006).  Most human cells do not feature telomerase, and thus their chromosomes lose telomere length with each cell replication.  As telomeres become too short to function, the cell will undergo the process of replicative senescence, where cell growth ceases.  The limited number of times a cell can divide before this occurs is determined by the telomere length of the cell that started the population (Baird et al., 2006), indicating that the length of the telomeres in the early embryo is extremely important in determining the number of times subsequent cells can divide.  It is important to understand how telomeres are kept at a functional length during the processes of spermatogenesis and embryogenesis, as these are the cells that are performing the most divisionsand would be prone to rapid telomere degradation.

Spermatogenesis

The activity of telomerase in the development of germ cells is vital to developing the telomeres to a length that will allow an embryo created with the germ cell to develop properly and have its cells divide a sufficient number of times.  Germ cells in both genders are developed from embryonic stem cells that are known as primordial germ cells (PGCs).  In males, the PGCs lie dormant after an initial period of proliferation until just after birth, when they begin to differentiate into spermatogonia (Coussens et al., 2006).  

The experiments performed by Coussens et al. demonstrated the role of TERT in telomerase activity during spermatogenesis.  They showed that the already well-established drop in telomerase activity during the quiescent period of developing PGCs is a result of lowered TERT expression (2006).  They tested 1500 purified primordial germ cells using Pou5f1-GFP transgenic mouse embryos that had been engineered to have fluorescent proteins in the PGCs to test for the presence of Tert mRNA, measured in comparison to Hprt1 levels, which indicates the level of TERT expression.  During days 10.5 and 12.5 of embryo development, while the PGCs are still developing, similar levels of Tert mRNA were found, but on days 15.5 and 16.5, when the PGCs are in a quiescent state, no Tert mRNA was found (Figure 1; Lake Forest College Users).  Further experimentation was performed to determine that TERT activity was not halted after translation from DNA.  A transgenic mouse strain using the chicken gene to ensure TERT expression (CAG-Tert strain) was created.  This strain shows a significantly greater amount of telomerase activity in comparison to non-transgenic embryos (Figure 2; Lake Forest College Users).  The homozygous CAG-Tert mice were then crossed with homozygous Pou5f1-GFP mice, using homozygous Pou5f1-GFP embryos as a control.  Coussens et al. found that not only did the transgenic embryos have about a 2.3 fold increase in telomerase activity when compared with control embryos (P < 0.01), but also that when the controls are quiescent and have no telomerase activity (days 15.5 and 16.5) the CAG-Tert transgenic PGCs still had telomerase activity (Figure 2; Lake Forest College Users) (2006).  Therefore, because separate expression of TERT restores telomerase activity, the primary inhibition process cannotbe post-translational.  A further experiment by Coussens et al. shows that though TERT is normally expressed before but not during quiescence; a TERT knockout strain that would have no TERT expression at any stage, or a CAG-Tert strain that would over-express TERT at all stages, all show no significant variation from the wild-type strain when measuring the frequency of cycling in PGCs (Figure 3; Lake Forest College Users).  These studies demonstrate that despite telomerase activity being controlled by the expression of TERT, the presence of extra telomerase or its complete absence will have no effect on the development and division of primordial germ cells (Coussens et al., 2006).  Even after the quiescent phase of the PGCs TERT expression has no effect on whether or not cells continue to differentiate into spermatozoa (Tanemura et al., 2005).  These mutated mouse strains will most likely have debilitating effects on future generations if they were allowed to breed, or if the mutation occurred in nature.  Future generations with the knockout gene for TERT will most likely face DNA degradation and chromosomal fusing.  In fact, it has been shown that mice mutant for the RNA component in telomerase (Terc^-/-) become infertile after the sixth generation (Tanemura et al., 2005).

Riou et al. performed a series of experiments to determine which specific cells feature telomerase activity in relation to spermatogenesis in adult mice; they attempt to discover when and where in spermatogenesis the telomeres are restored (2005).  The researchers isolated the SP fraction from the testis.  This population of cells contains spermatogonia and germinal stem cells.  Magnetic-activated cell sorting was used to select cells containing the á₆-integrin protein.  The á₆-integrin-positive Side Population (á₆⁺SP) cells were then tested to see if they contained Ep-CAM, an indicator of spermatogonia cells.  Ninety-five percent of the cells were seen to contain Ep-CAM (Figure 4; Lake Forest College Users).  The cells were then tested for the presence of CD9, another indicator of germinal stem cells and spermatogonia.  Ninety-seven percent of the á₆⁺SP stem cells displayed the CD9 marker (Figure 4; Lake Forest College Users).  The experimenters then performed assays that revealed telomerase activity in the á₆⁺SP stem cells to be greater than in the total extracts from the rest of the testis, indicating that telomerase activity is primarily in the á₆⁺SP stem cells of adult testis (Riou et al., 2005).  Further assays were performed to compare á₆⁺SP telomerase activity with telomerase activity in spermatocytes I, round and elongated spermatids, and epididymal spermatozoa (Figure 5; Lake Forest College Users).  Telomerase activity in the á₆⁺SP population was shown to be significantly greater than the activity in all the other tested groups, and that epididymal spermatozoa contain no telomerase activity at all.  These groups were tested in mice testes that were 2, 12, and 24 months old (Figure 5; Lake Forest College Users).  The experiment revealed no significant change in the telomerase activity in relation to age.  These experiments serve to show that telomere repair to create spermatozoa with sufficient length to produce healthy offspring occurs primarily in the á₆⁺SP cells, and that telomerase activity sharply drops as the sperm cells become more developed, and stops altogether in mature spermatozoa (Riou et al., 2005).