CHEK2 signaling is the key regulator of oocyte survival after chemotherapy.

Cancer treatments can damage the ovarian follicle reserve, leading to primary ovarian insufficiency and infertility among survivors. Checkpoint kinase 2 (CHEK2) deficiency prevents elimination of oocytes in primordial follicles in female mice exposed to radiation and preserves their ovarian function and fertility. Here, we demonstrate that CHEK2 also coordinates the elimination of oocytes after exposure to standard-of-care chemotherapy drugs. CHEK2 activates two downstream targets-TAp63 and p53-which direct oocyte elimination. CHEK2 knockout or pharmacological inhibition preserved ovarian follicle reserve after radiation and chemotherapy. However, the lack of specificity for CHEK2 among available inhibitors limits their potential for clinical development. These findings demonstrate that CHEK2 is a master regulator of the ovarian cellular response to damage caused by radiation and chemotherapy and warrant the development of selective inhibitors specific to CHEK2 as a potential avenue for ovario-protective treatments

SYCE2 is required for synaptonemal complex assembly, double strand break repair, and homologous recombination

Synapsis is the process by which paired chromosome homologues closely associate in meiosis before crossover. In the synaptonemal complex (SC), axial elements of each homologue connect through molecules of SYCP1 to the central element, which contains the proteins SYCE1 and -2. We have derived mice lacking SYCE2 protein, producing males and females in which meiotic chromosomes align and axes form but do not synapse. Sex chromosomes are unaligned, not forming a sex body. Additionally, markers of DNA breakage and repair are retained on the axes, and crossover is impaired, culminating in both males and females failing to produce gametes. We show that SC formation can initiate at sites of SYCE1/SYCP1 localization but that these points of initiation cannot be extended in the absence of SYCE2. SC assembly is thus dependent on SYCP1, SYCE1, and SYCE2. We provide a model to explain this based on protein–protein interactions

Mutation of the Mouse Syce1 Gene Disrupts Synapsis and Suggests a Link between Synaptonemal Complex Structural Components and DNA Repair

In mammals, the synaptonemal complex is a structure required to complete crossover recombination. Although suggested by cytological work, in vivo links between the structural proteins of the synaptonemal complex and the proteins of the recombination process have not previously been made. The central element of the synaptonemal complex is traversed by DNA at sites of recombination and presents a logical place to look for interactions between these components. There are four known central element proteins, three of which have previously been mutated. Here, we complete the set by creating a null mutation in the Syce1 gene in mouse. The resulting disruption of synapsis in these animals has allowed us to demonstrate a biochemical interaction between the structural protein SYCE2 and the repair protein RAD51. In normal meiosis, this interaction may be responsible for promoting homologous synapsis from sites of recombination

Sexual dimorphism in the meiotic requirement for PRDM9: A mammalian evolutionary safeguard.

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

Mouse HORMAD1 and HORMAD2, two conserved meiotic chromosomal proteins, are depleted from synapsed chromosome axes with the help of TRIP13 AAA-ATPase

Meiotic crossovers are produced when programmed double-strand breaks (DSBs) are repaired by recombination from homologous chromosomes (homologues). In a wide variety of organisms, meiotic HORMA-domain proteins are required to direct DSB repair towards homologues. This inter-homologue bias is required for efficient homology search, homologue alignment, and crossover formation. HORMA-domain proteins are also implicated in other processes related to crossover formation, including DSB formation, inhibition of promiscuous formation of the synaptonemal complex (SC), and the meiotic prophase checkpoint that monitors both DSB processing and SCs. We examined the behavior of two previously uncharacterized meiosis-specific mouse HORMA-domain proteins-HORMAD1 and HORMAD2-in wild-type mice and in mutants defective in DSB processing or SC formation. HORMADs are preferentially associated with unsynapsed chromosome axes throughout meiotic prophase. We observe a strong negative correlation between SC formation and presence of HORMADs on axes, and a positive correlation between the presumptive sites of high checkpoint-kinase ATR activity and hyper-accumulation of HORMADs on axes. HORMADs are not depleted from chromosomes in mutants that lack SCs. In contrast, DSB formation and DSB repair are not absolutely required for depletion of HORMADs from synapsed axes. A simple interpretation of these findings is that SC formation directly or indirectly promotes depletion of HORMADs from chromosome axes. We also find that TRIP13 protein is required for reciprocal distribution of HORMADs and the SYCP1/SC-component along chromosome axes. Similarities in mouse and budding yeast meiosis suggest that TRIP13/Pch2 proteins have a conserved role in establishing mutually exclusive HORMAD-rich and synapsed chromatin domains in both mouse and yeast. Taken together, our observations raise the possibility that involvement of meiotic HORMA-domain proteins in the regulation of homologue interactions is conserved in mammals

Expressions- und Funktionsanalyse der Keimzell-specifischen Gene für ADAM 27 und Testase 2

In den vorgelegten Arbeit wurden zwei Mitglieder der ADAM-Familie, ADAM27 und Testase 2 der Maus, untersucht. Beide Gene werden spezifisch in den testikulären Keimzellen exprimiert und deswegen können diesen Gene eine Rolle in der Spermatogenese und/oder beim Fertilisierungsprozess spielen. ADAM 27 ist ein putatives Adhäsionsprotein, und es wird vermutet, dass es an Integrine bindet, die Moleküle auf der Ooocytenoberfläche sind. Die Expression von ADAM27 wurde erstmals in den Spermatocyten detektiert. ADAM 27 findet sich zunächst im Golgi-Apparat und später hauptsächlich im Cytoplasma. Es ist nicht klar, ob das reife Protein während der Reifung der Spermien entfernt wird oder im Akrosom gespeichert wird. Die ersten Ergebnisse deuten jedoch darauf hin, dass ADAM 27 im epididymalen Spermienextrakt zu detektieren ist. Um die Funktion von ADAM 27 in der Spermatogenese und beim Fertilisationsprozess aufzuklären, wurden in vitro und in vivo Ansätze gewählt. Im in vitro Ansatz wurde mithilfe von in vitro sythetisierten rekombinanten ADAM 27-Polypeptiden mit verschiedenen funktionellen Regionen (Disintegrin, Cys-reich, EGF-like) die Rolle des Proteins bei der Spermien-Oocyten Bindung untersucht. Die Ergebnisse aus diesen Experimenten weisen darauf hin, dass ADAM 27 durch die Cys-reiche Domäne an die Zona pellucida von Oocyten bindet. Für die funktionellen Untersuchung von ADAM 27 in vivo, wurde zwei Knockout Konstrukte hergestellt. Bei dem ersten Konstrukt wurde die Transmembrandomäne deletiert. Die homozygoten mänlichen und weiblichen Tiere sind fertil und zeigen keine Abnormalitäten ausser einer reduzierten Spermienmotilität. Bei dem zweiten Konstrukt wurden die ersten drei Exons (mit ATG) deletiert, um die Synthese des gesamten Proteins zu verhindern. Keine der vier produzierten Chimären zeigten Keimbahntransmission, was auf eine Haploinsuffizienz hinweist. Drei von vier Chimären mit über 70% Chimärismus waren infertil. Dies deutet darauf hin, dass ADAM 27 für eine normale Keimzellentwicklung von Bedeutung ist. Das zweite untersuchte Gen war Testase 2, das auch als ADAM 25 bekannt ist. In der Literatur wurde Testase 2 als single copy Gen identifiziert. Unsere Untersuchungen haben aber gezeigt, dass es zwei Kopien des Gens auf Chromosom 8 gibt. Es wurde festgestellt, dass die beiden Gene im selben Spermatogenese-Stadium exprimiert werden. Aus diesem Grund wurden die Experimente für die funktionelle Analyse von Testase 2 in Knockout Mäusen eingestellt. Um die Funktion des Testase 2 Gens aufzuklären, müssen andere Strategien entwickelt werden, in denen die beiden Kopien dieses Gens ausgeschaltet werden

Meiosis: the chromosomal foundation of reproduction.

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

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

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\u27s 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

A mouse geneticist\u27s practical guide to CRISPR applications.

CRISPR/Cas9 system of RNA-guided genome editing is revolutionizing genetics research in a wide spectrum of organisms. Even for the laboratory mouse, a model that has thrived under the benefits of embryonic stem (ES) cell knockout capabilities for nearly three decades, CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 technology enables one to manipulate the genome with unprecedented simplicity and speed. It allows generation of null, conditional, precisely mutated, reporter, or tagged alleles in mice. Moreover, it holds promise for other applications beyond genome editing. The crux of this system is the efficient and targeted introduction of DNA breaks that are repaired by any of several pathways in a predictable but not entirely controllable manner. Thus, further optimizations and improvements are being developed. Here, we summarize current applications and provide a practical guide to use the CRISPR/Cas9 system for mouse mutagenesis, based on published reports and our own experiences. We discuss critical points and suggest technical improvements to increase efficiency of RNA-guided genome editing in mouse embryos and address practical problems such as mosaicism in founders, which complicates genotyping and phenotyping. We describe a next-generation sequencing strategy for simultaneous characterization of on- and off-target editing in mice derived from multiple CRISPR experiments. Additionally, we report evidence that elevated frequency of precise, homology-directed editing can be achieved by transient inhibition of the Ligase IV-dependent nonhomologous end-joining pathway in one-celled mouse embryos. Genetics 2015 Jan; 199(1):1-15

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 entering meiotic prophase I are eliminated during Fetal Oocyte Attrition (FOA) and the first week of postnatal life. Three major mechanisms regulate the number of oocytes that survive during development and establish the ovarian reserve in females entering puberty. In the first wave of oocyte loss, 30-50% of the oocytes are eliminated during early FOA, a phenomenon that is attributed to high Long interspersed nuclear element-1 (LINE-1) expression. The second wave of oocyte loss is the elimination of oocytes with meiotic defects by a meiotic quality checkpoint. The third wave of oocyte loss occurs perinatally during primordial follicle formation when some oocytes fail to form follicles. It remains unclear what regulates each of these three waves of oocyte loss and how they shape the ovarian reserve in either mice or humans. Immunofluorescence and 3D visualization have opened a new avenue to image and analyze oocyte development in the context of the whole ovary rather than in less informative 2D sections. This article provides a comprehensive protocol for whole ovary immunostaining and optical clearing, yielding preparations for imaging using multiphoton microscopy and 3D modeling using commercially available software. It shows how this method can be used to show the dynamics of oocyte attrition during ovarian development in C57BL/6J mice and quantify oocyte loss during the three waves of oocyte elimination. This protocol can be applied to prenatal and early postnatal ovaries for oocyte visualization and quantification, as well as other quantitative approaches. Importantly, the protocol was strategically developed to accommodate high-throughput, reliable, and repeatable processing that can meet the needs in toxicology, clinical diagnostics, and genomic assays of ovarian function