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Whole Ovary Immunofluorescence, Clearing, and Multiphoton Microscopy for Quantitative 3D Analysis of the Developing Ovarian Reserve in Mouse

Female fertility and reproductive lifespan depend on the quality and quantity of the ovarian oocyte reserve. An estimated 80% of female germ cells ...

The Jackson Laboratory

The transcriptional regulation of mammalian meiosis is poorly characterized, owing to few genetic and ex vivo models. From a genetic screen, we identify the transcription factor MYBL1 as a male-specific master regulator of several crucial meiotic processes. Spermatocytes bearing a novel separation-of-function allele (Mybl1(repro9)) had subtle defects in autosome synapsis in pachynema, a high incidence of unsynapsed sex chromosomes, incomplete double-strand break repair on synapsed pachytene chromosomes and a lack of crossing over. MYBL1 protein appears in pachynema, and its mutation caused specific alterations in expression of diverse genes, including some translated postmeiotically. These data, coupled with chromatin immunoprecipitation (ChIP-chip) experiments and bioinformatic analysis of promoters, identified direct targets of MYBL1 regulation. The results reveal that MYBL1 is a master regulator of meiotic genes that are involved in multiple processes in spermatocytes, particularly those required for cell cycle progression through pachynema.

A-MYB (MYBL1) transcription factor is a master regulator of male meiosis.

Telomere-led rapid prophase movements (RPMs) in meiotic prophase have been observed in diverse eukaryote species. A shared feature of RPMs is that the force that drives the chromosomal movements is transmitted from the cytoskeleton, through the nuclear envelope, to the telomeres. Studies in mice suggested that dynein movement along microtubules is transmitted to telomeres through SUN1/KASH5 nuclear envelope bridges to generate RPMs. We monitored RPMs in mouse seminiferous tubules using 4D fluorescence imaging and quantitative motion analysis to characterize patterns of movement in the RPM process. We find that RPMs reflect a combination of nuclear rotation and individual chromosome movements. The telomeres move along microtubule tracks that are apparently continuous with the cytoskeletal network and exhibit characteristic arrangements at different stages of prophase. Quantitative measurements confirmed that SUN1/KASH5, microtubules, and dynein, but not actin, were necessary for RPMs and that defects in meiotic recombination and synapsis resulted in altered RPMs.

Mechanism and regulation of rapid telomere prophase movements in mouse meiotic chromosomes.

Of mice and CRISPR: The post-CRISPR future of the mouse as a model system for the human condition.

Ovarian function is directly correlated with survival of the primordial follicle reserve. Women diagnosed with cancer have a primary imperative of treating the cancer, but since the resting oocytes are hypersensitive to the DNA-damaging modalities of certain chemo- and radiotherapeutic regimens, such patients face the collateral outcome of premature loss of fertility and ovarian endocrine function. Current options for fertility preservation primarily include the collection and cryopreservation of oocytes or -fertilized oocytes, but this necessitates a delay in cancer treatment and additional assisted reproductive technology procedures. Here, we evaluated the potential of pharmacological preservation of ovarian function by inhibiting a key element of the oocyte DNA damage checkpoint response, checkpoint kinase 2 (CHK2; CHEK2). Whereas nonlethal doses of ionizing radiation (IR) eradicate immature oocytes in wild-type mice, irradiated  mice retain their oocytes and, thus, fertility. Using an ovarian culture system, we show that transient administration of the CHK2 inhibitor 2-(4-(4-chlorophenoxy)phenyl)-H-benzimidazole-5-carboxamide-hydrate ("CHK2iII") blocked activation of the CHK2 targets TRP53 and TRP63 in response to sterilizing doses of IR, and preserved oocyte viability. After transfer into sterilized host females, these ovaries proved functional and readily yielded normal offspring. These results provide experimental evidence that chemical inhibition of CHK2 is a potentially effective treatment for preserving the fertility and ovarian endocrine function of women exposed to DNA-damaging cancer therapies such as IR.

Pharmacological Inhibition of the DNA Damage Checkpoint Prevents Radiation-Induced Oocyte Death.

The survival rate of reproductive-age patients with cancer is increasing, reflecting the advent of better and more efficient therapies. Cancer survivors seek the resumption of a normal and healthy life, which often includes starting a family. Unfortunately, many cancer treatments increase the risk of premature ovarian insufficiency (POI) and infertility. Assisted reproductive technologies (ART) can address infertility, but fail to preserve the natural function of the ovaries as a source of hormones that regulate many aspects of women's health. The advancement of fertoprotective technologies is hindered by our lack of understanding of oocyte biology and their sensitivity to cancer therapies. Because many cancer treatments cause DNA damage, apoptosis is thought to be the major mechanism eliminating damaged oocytes. Indeed, recent studies in mice demonstrate that targeting proteins involved in apoptosis protects oocytes and prevents infertility in females exposed to radiation. Therefore, a better appreciation of oocyte response to radiation and anticancer drugs will uncover new targets for the development of specialized therapies to prevent ovarian failure. We make a case here for the necessity of such fertoprotective treatments. We review recent findings that have significantly advanced our understanding of how cancer therapies induce apoptotic death in oocytes, and how we could use this knowledge to design better fertoprotective treatments.

Prolonging Reproductive Life after Cancer: The Need for Fertoprotective Therapies.

Pairing and synapsis of homologous chromosomes during meiosis is crucial for producing genetically normal gametes and is dependent upon repair of SPO11-induced double-strand breaks (DSBs) by homologous recombination. To prevent transmission of genetic defects, diverse organisms have evolved mechanisms to eliminate meiocytes containing unrepaired DSBs or unsynapsed chromosomes. Here we show that the CHK2 (CHEK2)-dependent DNA damage checkpoint culls not only recombination-defective mouse oocytes but also SPO11-deficient oocytes that are severely defective in homolog synapsis. The checkpoint is triggered in oocytes that accumulate a threshold level of spontaneous DSBs (∼10) in late prophase I, the repair of which is inhibited by the presence of HORMAD1/2 on unsynapsed chromosome axes. Furthermore, Hormad2 deletion rescued the fertility of oocytes containing a synapsis-proficient, DSB repair-defective mutation in a gene (Trip13) required for removal of HORMADs from synapsed chromosomes, suggesting that many meiotic DSBs are normally repaired by intersister recombination in mice.

The DNA Damage Checkpoint Eliminates Mouse Oocytes with Chromosome Synapsis Failure.

Meiosis is the chromosomal foundation of reproduction, with errors in this important process leading to aneuploidy and/or infertility. In this review celebrating the 50th anniversary of the founding of the Society for the Study of Reproduction, the important chromosomal structures and dynamics contributing to genomic integrity across generations are highlighted. Critical unsolved biological problems are identified, and the advances that will lead to their ultimate resolution are predicted.

Meiosis: the chromosomal foundation of reproduction.

In many mammals, genomic sites for recombination are determined by the histone methyltransferase PRMD9. Some mouse strains lacking PRDM9 are infertile, but instances of fertility or semifertility in the absence of PRDM9 have been reported in mice, canines, and a human female. Such findings raise the question of how the loss of PRDM9 is circumvented to maintain fertility. We show that genetic background and sex-specific modifiers can obviate the requirement for PRDM9 in mice. Specifically, the meiotic DNA damage checkpoint protein CHK2 acts as a modifier allowing female-specific fertility in the absence of PRDM9. We also report that, in the absence of PRDM9, a PRDM9-independent recombination system is compatible with female meiosis and fertility, suggesting sex-specific regulation of meiotic recombination, a finding with implications for speciation.

Ewelina Bolcun-Filas

The Jackson Laboratory

Whole Ovary Immunofluorescence, Clearing, and Multiphoton Microscopy for Quantitative 3D Analysis of the Developing Ovarian Reserve in Mouse