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What makes a good egg?
In approximately 15 percent of cases where couples are unable to conceive, the underlying cause remains unknown. Now, research at University of California San Diego School of Medicine, Division of Biological Sciences has identified ZFP36L2 — L2 for short — a protein in mice that has to be present in eggs for them to complete normal development. Without it eggs appear normal, but cannot be fertilized by sperm. Female mice lacking L2 in their eggs ovulate and are otherwise healthy, but produce no offspring.
As humans also have the L2 protein, this finding, published February 5 in Developmental Cell, gives research a new place to look for answers to these currently unexplained causes of female infertility.
Explains senior author Heidi Cook-Andersen MD PhD, assistant professor of reproductive medicine and biological sciences at UC San Diego, and physician at the UC San Diego-affiliated Reproductive Partners Fertility Center-San Diego: "In my clinical practice, I work with couples struggling with infertility. All of their tests come back normal. But, there are many things important for fertility that we still haven't discovered and we can't test for what we don't know. That's why this study is so exciting to us. It's another clue."
To answer this question, Cook-Andersen and her team worked with female mice engineered to completely lack the L2 protein in their eggs. The protein continued to function normally in all other parts of the mouse body.
To determine what effect this had on fertility, Cook-Andersen's team set up fertile male mice with 10 females lacking L2 in their eggs, and 10 normal females. Then they tracked the groups for six months. In that time, normal mice produced regularly, giving birth to a litter of seven or eight pups approximately every 21 days, having a total of about 60 baby mice.
Female mice lacking L2 protein in their eggs didn't produce a single pup.
Looking deeper, they found L2-deficient eggs were unable to undergo global transcription silencing, a process that normally happens in the final stages of egg growth. During that stage, transcription - the conversion of encoded gene "recipes" from DNA into mRNA (which carries the recipe to the cell's protein making machinery) - is completely shut down. It's an important step in animals from worms to humans. Eggs remain in this "quiet" state until they are fertilized and begin developing into an embryo. However, eggs without L2 continue transcribing genes into mRNA and producing proteins.
Cook-Andersen believes the mouse infertility was due at least in part to their failure to enter global transcription silencing. It may also be possible L2 affects other factors influencing fertility as yet unidentified.
Beyond their potential significance to human infertility, the results are exciting because they identify that in the egg, L2 degrades mRNAs based on protein marks intimately associated with DNA. This is a clue to how L2 and RNA decay might shut off transcription. Previously, not much was known about how or when this crucial developmental event occurs in eggs.
"Many research groups are looking at how genes are regulated from the perspective of which genes need to be turned 'on' for a cell to advance to the next stage of development, but now we see that it's just as important to know which genes need to be turned 'off.'"
In the future, the team plans to determine if L2 plays a role in human infertility. In the meantime, they are using their mouse model to learn more about the steps needed for global transcriptional silencing and to identify additional factors required to make a good egg.
• Oocyte global transcriptional silencing is mediated by an mRNA decay activator
• ZFP36L2 downregulates master regulators of transcription during oocyte growth
• ZFP36L2 binds and degrades a group of mRNAs encoding demethylases for H3K4 and H3K9
• ZFP36L2 enables increased histone methylation associated with transcription silencing
Global transcriptional silencing is a highly conserved mechanism central to the oocyte-to-embryo transition. We report the unexpected discovery that global transcriptional silencing in oocytes depends on an mRNA decay activator. Oocyte-specific loss of ZFP36L2 an RNA-binding protein that promotes AU-rich element-dependent mRNA decay prevents global transcriptional silencing and causes oocyte maturation and fertilization defects, as well as complete female infertility in the mouse. Single-cell RNA sequencing revealed that ZFP36L2 downregulates mRNAs encoding transcription and chromatin modification regulators, including a large group of mRNAs for histone demethylases targeting H3K4 and H3K9, which we show are bound and degraded by ZFP36L2. Oocytes lacking Zfp36l2 fail to accumulate histone methylation at H3K4 and H3K9, marks associated with the transcriptionally silent, developmentally competent oocyte state. Our results uncover a ZFP36L2-dependent mRNA decay mechanism that acts as a developmental switch during oocyte growth, triggering wide-spread shifts in chromatin modification and global transcription.
Authors: Jennifer N. Dumdie, Kyucheol Cho, Madhuvanthi Ramaiah, David Skarbrevik, Sergio Mora-Castilla, Deborah J. Stumpo, Jens Lykke-Andersen, Louise C. Laurent, Perry J. Blackshear, Miles F. Wilkinson, Heidi Cook-Andersen
Co-authors also include: Jennifer N. Dumdie, Kyucheol Cho, Madhuvanthi Ramaiah, David Skarbrevik, Sergio Mora-Castilla, Jens Lykke-Andersen, Louise C. Laurent, Miles F. Wilkinson, UC San Diego; Deborah F. Stumpo, National Institute of Environmental Health Sciences; and Perry F. Blackshear, National Institute of Environmental Health Sciences and Duke University Medical Center.
Keywords: ZFP36L2, oocyte developmental competence, AU-rich element, RNA decay, transcriptional silencing, histone methylation, H3K4, H3K9, histone demethylase, female fertility.
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Single-cell RNA sequencing reveals how protein L2 turns off mRNA's ability to encode histone methylation. Oocytes without L2 fail to enter into a "quiet state." Therefore, L2 must act as a developmental switch in an oocyte, that triggers a global transcription shutdown in order for an egg to become less energiaed and allow fertilization to occur. Image credit: Heidi Cook-Andersen lab.