The Role of LINE-1 in the Death of a Fetal Oocyte

By A.K. Matai

Fetal oocyte attrition is a poorly understood process in which approximately 80% of meiotic prophase I oocytes are eliminated through apoptotic pathways before birth1, 2. Fetal oocyte attrition is not unique to humans as it has been observed in primates as well as invertebrates3, 4. However, the primary triggers of fetal oocyte attrition are unknown. One hypothesis that has been proposed to explain this phenomenon is called “death by defect” which suggests that defective oocytes are preferentially eliminated in an effort to retain competent oocytes for the formation of primordial follicles5. In this paper, Malki et. al. uncovered a novel role of the LINE-1 retrotransposon that could potentially act as a trigger for apoptotic pathways in fetal oocytes leading to fetal oocyte attrition.

Currently, the LINE-1 retrotransposon comprises 20% of the human genome and is the only active transposable elements in humans (Figure 1)6. The LINE-1 retrotransposon is effectively silenced using DNA methylation as well as PIWI-interacting RNAs (piRNA) which prevent its mobilization using reverse transcription7, 8. Recently, it has become clear the epigenetic reset of DNA methylation during embryogenesis provides a window where the repression of the LINE-1 retrotransposon is lifted. The LINE-1 retrotransposon is active when the primordial germ cells are entering the meiotic prophase I of oogenesis (Figure 3). This poses a threat to the germline genome as the LINE-1 retrotransposon could potentially be inserted into a critical genomic region (Figure 2). The authors of this study set out to determine how the activity of LINE-1 retrotransposon affects the viability as well as quality of the fetal oocyte.

To explore this, Malki et. al. examined the levels of LINE-1 mRNA and LINE-1 ORF1 protein in wild type oocytes as well as maelstrom null mutants. The maelstrom homolog gene in mice encodes the Mael protein that is a key player in piRNA-mediated transposon silencing. Therefore, the maelstrom null mutants would have elevated levels of LINE-1 activity as the LINE-1 retrotransposon could no longer be repressed9. They noticed that there was an approximate 2-fold increase in LINE-1 mRNA and LINE-1 ORF1 protein levels compared to the wild type controls. Not only that but the maelstrom null mutants showed increased signs of fetal oocyte attrition when compared to the wild type control indicating that the upregulation of LINE-1 retrotransposon activity does cause a decrease in oocyte viability.

But that begs the question, when you lift the repression on the LINE-1 retrotransposon, why do only some oocytes die as opposed to all of them? To address this question, the authors used immunofluorescence to characterize relative mean nuclear levels of the LINE-1 ORF1 protein (RMN units) in individual fetal oocytes at developmental stages E15.5 (embryonic day 15.5), E18.5 (embryonic day 18.5) and P2 (postnatal day 2). Surprisingly, the authors found that there was quite a bit of variability in the amounts of LINE-1 ORF1 protein being expressed in individual fetal oocytes. Another important observation that emerges from this experiment is that the level of LINE-1 ORF1 protein expressed at E15.5 determined the chances of fetal oocyte survival at E18.5 and P2. Suppose a fetal oocyte expressed 1.5 RMN units at E15.5 and the threshold at E18.5 and P2 was 1.6 and 1.4 RMN units respectively. That particular oocyte would survive past E18.5 as its RMN value was under the threshold but it will be killed at P2 as its RMN values exceeds the threshold. Further examination of the fetal oocytes in maelstrom null mutants revealed that excessive LINE-1 expression resulted in a higher proportion of DNA double stranded breaks that were not being repaired by MCHI, a mismatch repair protein, as well as synapsis failure of homologous chromosomes during meiotic prophase I. Overall, recombination was impaired, the fetal oocytes were showing meiotic abnormalities and genes involved in apoptotic pathways were upregulated suggesting the LINE-1 activity was acting as the trigger to induce fetal oocyte attrition.

To corroborate the results seen in the maelstrom null mutants, the authors used an ORFeus transgene that was composed of a tetracycline induced LINE-1 element. Since there is already a high background expression of LINE-1 as there are 11 000 full-length endogenous LINE-1 elements in the mouse genome10, the effect of the ORFeus transgene was mild. Regardless, the ORFeus transgene fetal ovaries had fewer oocytes and increased levels of unrepaired DNA damage and asynapsis (the failure of homologous chromosomes to properly pair during meiosis). The gain of function experiment involving the ORFeus transgene verified the results seen in the loss of function experiments involving maelstrom null mutants.

To test their hypothesis that LINE-1 activity results in an increase in fetal oocyte attrition, Malki et. al. reasoned that inhibiting the activity of LINE-1 should reduce fetal oocyte attrition. To do this, the authors gave wild type as well as maelstrom null mutant pregnant female mice azidothymidine (AZT). AZT acts as an inhibitor of reverse transcriptases including the LINE-1 ORF2 protein resulting in the inability of the LINE-1 retrotransposon to mobilize12. The AZT treated wild type ovaries retained 96% of its starting oocyte population at E18.5 whereas the AZT treated maelstrom null mutant ovaries retained 61% of its starting oocyte population at E18.5 showing that inhibiting LINE-1 activity is sufficient to reduce fetal oocyte attrition in mice ovaries. However, at P2, ATZ treated wild type and AZT treated maelstrom null mutant ovaries retained only 41% and 31% of their starting oocyte populations (Figure 3). But this was not unexpected as AZT does not inhibit the endonuclease activity of LINE-1 ORF2 protein, still allowing it to cause double stranded DNA breaks.

From these set of experiments, Malki et. al. propose the following model (Figure 3). During embryonic reprogramming, the DNA methylation repression is lifted from the LINE-1 retrotransposon elements. LINE-1 creates a reverse transcriptase dependent intermediate that causes DNA double stranded breaks, impedes the repair of the breaks impeding recombination and also causes meiotic pairing abnormalities. This triggers the apoptotic pathways in the oocyte leading to its death.

Malki et. al.’s paper highlights the role of LINE-1 retrotransposons in fetal oocyte attrition. But as with many discoveries, their research unearths many more questions. In somatic cells, transposon expression is upregulated in response to cellular stimuli or stress such as heat shock11. It would be worthwhile to explore if similar stimuli cause upregulate LINE-1 retrotransposons in fetal oocyte cells. From a practical perspective, the suppression of oocyte attrition through the use of AZT might present a unique opportunity to extend the female reproductive lifespan – a potential avenue of fertility treatment for women who may choose to have kids at a later age.

 the-role-of-line-1-in-the-death-of-a-fetal-oocyte-figure-1

Figure 1. Overview of LINE-1 Mobilization and Integration into the Genome. The active LINE-1 (L1) element contains two open reading frames, ORF1 and ORF2. L1 is transcribed in nucleus by RNA polymerase II and then transported into the cytoplasm. There, ORF1 and ORF2 are translated. Both proteins bind the L1 mRNA forming a ribonucleoprotein complex which is transported into the nucleus. ORF2p contains an endonuclease domain as well as a reverse transcriptase allowing it to cleave a potential target site and begin cDNA synthesis for L1 integration. The integrated L1 element is flanked by the target site duplication.

 the-role-of-line-1-in-the-death-of-a-fetal-oocyte-figure-2

Figure 2.  Potential Consequences of LINE-1 Genome Integration. [A] Illustration of a LINE-1 element and an arbitrary gene. 5’ and 3’ UTR are represented by the dark blue boxes, the coding exons are represented by the light blue boxes and the splicing of exons into mRNA are indicated by the broken lines. [B] LINE-1 can insert into an exon leading to a disruption in the open reading frame leading to a modified or truncated protein. [C] LINE-1 insertion into an intron can have deleterious effects such as the creation of a new exon or an alteration of the mRNA splicing pattern. [D] LINE-1 elements can promote inversions within the genome. [E] The LINE-1 promoter can influence the transcription of neighbouring genes.

the-role-of-line-1-in-the-death-of-a-fetal-oocyte-figure-3

 

Figure 3. Overview of Malki et. al. Findings in  “A role for retrotransposon LINE-1 in fetal oocyte attrition in Mice”. [A] Figure adapted from Malki et. al. that illustrates the window of LINE-1 activation in red. [B] Wild type and maelstrom null pregnant female mice were treated with AZT, a reverse transcriptase inhibitor. The wild type oocytes without AZT treatment showed that 55% of the original number of oocytes were retained at E15.5 whereas treatment with AZT resulted in the retention of 96% of the original number of oocytes at E15.5. Both wild type oocytes with and without AZT treatment only retained 41% and 40% of the original number of oocytes respectively at P2. The maelstrom null oocytes without AZT treatment retained 18% of the original number of oocytes at E15.5 whereas treatment with AZT resulted in the retention of 61% of the original number of oocytes. Both maelstrom null oocytes with and without AZT treatment only retained 31% and 14% of the original number of oocytes respectively at P2. [C] High levels of LINE-1 activity result in an increase in DNA damage as well as asynapsis in an oocyte. This results in the activation of apoptotic pathways and eventual fetal oocyte death. On the other hand, low levels of LINE-1 activity show low levels of DNA damage and asynapsis resulting in the survival of these oocytes.

References 

  1. Baker, T. G. (1963). A quantitative and cytological study of germ cells in human ovaries.Proceedings of the royal society of london. Series b, biological sciences, 417-433.
  2. Kurilo, L. F. (1981). Oogenesis in antenatal development in man.Human genetics57(1), 86-92.
  3. Beaumont, H. M. & Mandl, A. M. (1961). A quantitative and cytological study of oogonia and oocytes in the foetal and neonatal rat. R. Soc. Lond. B 155, 557–579.
  4. Matova, N., & Cooley, L. (2001). Comparative aspects of animal ogenesis. Developmental biology,231(2), 291-320.
  5. Tilly, J. L. (2001). Commuting the death sentence: how oocytes strive to survive.Nature Reviews Molecular Cell Biology2(11), 838-848.
  6. Goodier, J. L., & Kazazian, H. H. (2008). Retrotransposons revisited: the restraint and rehabilitation of parasites.Cell135(1), 23-35.
  7. Aravin, A. A., Van Der Heijden, G. W., Castañeda, J., Vagin, V. V., Hannon, G. J., & Bortvin, A. (2009). Cytoplasmic compartmentalization of the fetal piRNA pathway in mice.PLoS Genet5(12), e1000764.
  8. Babushok, D. V., & Kazazian, H. H. (2007). Progress in understanding the biology of the human mutagen LINE‐Human mutation28(6), 527-539.
  9. Sato, K., & Siomi, M. C. (2015). Functional and structural insights into the piRNA factor Maelstrom.FEBS letters589(14), 1688-1693.
  10. Goodier, J. L., Ostertag, E. M., Du, K., & Kazazian, H. H. (2001). A novel active L1 retrotransposon subfamily in the mouse.Genome research11(10), 1677-1685.
  11. Vasilyeva, L. A., Bubenshchikova, E. V., & Ratner, V. A. (1999). Heavy heat shock induced retrotransposon transposition in Drosophila.Genetical research74(02), 111-119.
  12. Dai, L., Huang, Q., & Boeke, J. D. (2011). Effect of reverse transcriptase inhibitors on LINE-1 and Ty1 reverse transcriptase activities and on LINE-1 retrotransposition.BMC biochemistry12(1), 1.
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