CHEK2 SIGNALING IS THE KEY REGULATOR OF OOCYTE SURVIVAL AFTER CHEMOTHERAPY

Radiation and chemotherapy can damage the primordial follicle reserve in female cancer patients leading to ovarian failure and infertility. Preservation of ovarian function requires treatment strategies that prevent loss of immature oocytes in primordial follicles during cancer therapy. Checkpoint kinase 2 (CHEK2) inhibition prevents loss of primordial oocytes caused by DNA damage and thus is a promising target for ovoprotective treatment against genotoxic agents. To determine which cancer treatments could benefit from ovoprotective activity of CHEK2 inhibition we investigated oocyte survival in Chek2-/- mice exposed to different chemotherapy drugs. Here, we show that loss of CHEK2 function prevents elimination of primordial oocytes damaged by cisplatin, cyclophosphamide, mafosfamide, doxorubicin, and etoposide, suggesting it could be used to reduce ovarian damage caused by wide range of drugs. Using genetic knockouts we reveal a critical role for TRP53 in oocyte response to chemotherapy drugs and show that both targets of CHEK2, TAp63 and TRP53, are activated by cisplatin and cyclophosphamide. Furthermore, we show that checkpoint kinase inhibitor and radiation- and chemotherapy sensitizer AZD7762 reduces oocyte elimination after radiation and chemotherapy treatments, despite its cytotoxic effect on ovarian somatic cells. Altogether, these findings demonstrate the role for CHEK2 as the master regulator of primordial oocyte survival or death and credential its targeting for ovoprotective treatments.

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Follistatin Regulates Germ Cell Nest Breakdown and Primordial Follicle Formation

Endocrinology ◽

10.1210/en.2010-0950 ◽

2010 ◽

Vol 152 (2) ◽

pp. 697-706 ◽

Cited By ~ 35

Author(s):

Fuminori Kimura ◽

Lara M. Bonomi ◽

Alan L. Schneyer

Keyword(s):

Germ Cell ◽

Germ Cells ◽

Ovarian Function ◽

Primordial Follicle ◽

Biochemical Properties ◽

The Body ◽

Protein Isoforms ◽

Primordial Follicles ◽

Reduced Rate ◽

Regulatory Functions

Abstract Follistatin (FST) is an antagonist of activin and related TGFβ superfamily members that has important reproductive actions as well as critical regulatory functions in other tissues and systems. FST is produced as three protein isoforms that differ in their biochemical properties and in their localization within the body. We created FST288-only mice that only express the short FST288 isoform and previously reported that females are subfertile, but have an excess of primordial follicles on postnatal day (PND) 8.5 that undergo accelerated demise in adults. We have now examined germ cell nest breakdown and primordial follicle formation in the critical PND 0.5–8.5 period to test the hypothesis that the excess primordial follicles derive from increased proliferation and decreased apoptosis during germ cell nest breakdown. Using double immunofluorescence microscopy we found that there is virtually no germ cell proliferation after birth in wild-type or FST288-only females. However, the entire process of germ cell nest breakdown was extended in time (through at least PND 8.5) and apoptosis was significantly reduced in FST288-only females. In addition, FST288-only females are born with more germ cells within the nests. Thus, the excess primordial follicles in FST288-only mice derive from a greater number of germ cells at birth as well as a reduced rate of apoptosis during nest breakdown. These results also demonstrate that FST is critical for normal regulation of germ cell nest breakdown and that loss of the FST303 and/or FST315 isoforms leads to excess primordial follicles with accelerated demise, resulting in premature cessation of ovarian function.

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Structural basis for small molecule targeting of Doublecortin Like Kinase 1 DCLK1

10.21203/rs.3.rs-133153/v1 ◽

2021 ◽

Author(s):

Onisha Patel ◽

Michael Roy ◽

Ashleigh Kropp ◽

Weiwen Dai ◽

Isabelle Lucet

Keyword(s):

Kinase Inhibitor ◽

Critical Role ◽

Structural Data ◽

Kinase Domain ◽

Microtubule Assembly ◽

Systematic Evaluation ◽

Structural Basis ◽

Allosteric Site ◽

Functional Protein ◽

Wide Range

Abstract Doublecortin-like kinase 1 (DCLK1) is a bi-functional protein classified as a Microtubule-Associated Protein (MAP) and as a serine/threonine kinase that plays a critical role in regulating microtubule assembly. This understudied kinase is upregulated or mutated in a wide range of cancers. Knockdown studies have shown that DCLK1 is functionally important for tumour growth. However, the presence of tissue and development specific spliced DCLK1 isoforms and the lack of systematic evaluation of their biological function have challenged the development of effective strategies to understand the role of DCLK1 in oncogenesis. Recently, DCLK1-IN-1 was reported as a potent and selective DCLK1 kinase inhibitor, a powerful new tool to dissect DCLK1 biological functions. Here, we report the crystal structures of DCLK1 kinase domain in complex with two DCLK1-IN-1 precursors and DCLK-IN-1. Combined, our structural data analysis illuminates and rationalises the structure-activity relationship that informed development of DCLK1-IN-1 and provides the basis for DCLK1-IN-1 increased selectivity. We show that DCLK1-IN-1 induces a drastic conformational change of the N-lobe, which uncovered a new allosteric site. In addition, we demonstrate that DCLK1-IN-1 binds DCLK1 long isoforms with high affinity but does not prevent DCLK1 MAP function. Together, our work outlines the need for in-depth studies to rationally design of isoform-specific modulators and provides an invaluable structural platform to further the design of selective DCLK1 therapeutic agents.

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A Shoreline Response Program (SRP) Decision Support Tool based on the Geographic Variability in Attenuation and Weathering of Stranded Oil.

International Oil Spill Conference Proceedings ◽

10.7901/2169-3358-2021.1.1141234 ◽

2021 ◽

Vol 2021 (1) ◽

Author(s):

Ed Owens ◽

Elliott Taylor ◽

Chunjiang An ◽

Zhi Chen ◽

George Danner ◽

...

Keyword(s):

Decision Support ◽

Web Application ◽

Treatment Options ◽

Critical Role ◽

Treatment Strategies ◽

Decision Support Tool ◽

Data Bases ◽

Support Tool ◽

Fine Grained ◽

Wide Range

ABSTRACT #1141234 The coastal waters of Canada embrace a wide range of physical environments and ecosystems from the warm, sediment-rich waters of the Bay of Fundy to the nutrient-limited cold waters of the high Arctic. This range of biophysical characteristics impacts natural attenuation and weathering processes for oil stranded on shorelines. This study was conducted to: 1) identify and quantify the primary regional parameters that control shoreline oil translocation (removal) processes and pathways and 2) define the effectiveness and environmental consequences of current and potential oiled shoreline treatment strategies and tactics. A specific knowledge gap, here and elsewhere in the world, has been in understanding how the distribution and character of fine-grained sediments affect stranded oil attenuation. Fine-grained sediments (<1mm) can play a critical role in natural or induced (that is, shoreline treatment) oil dispersal. Shoreline sediment samples were collected and analyzed from representative locations on Arctic, Atlantic, and Pacific Ocean beaches to provide a broad geographic characterization of mineral fines at the regional level. This knowledge is the basis for an “Oiled Shoreline Response Program (SRP) Decision Support Tool” to aid spill scientists, students, environmental resource managers, spill responders and the public in understanding the response methods and the ramifications and consequences of their shoreline treatment options without the need to digest technical papers, large reports, or data bases. This MPRI SRP Decision Support Tool is intended to be a dynamic, interactive, multi-layered, geographically and seasonally-based model for shoreline oil spill response decision analyses. A goal of this interactive model is to move away from the traditional static format of learning from explanations in text reports and publications to an interactive tool that encourages its users to explore and fully understand the significance of the different environmental factors outlined in publications and data bases. Recent advances in web technology make this possible. The development of user interface platforms such as React, libraries such as D3, and notebook forms like Observable has created a palette of technologies that together make web application patterns such as Documodels a much more streamlined development process. The power of this medium is to convey a complex subject and to enable a user to grasp keen insights and so understand the consequences of intervention decisions.

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Metformin: a novel promising option for fertility preservation during cyclophosphamide-based chemotherapy

MHR Basic science of reproductive medicine ◽

10.1093/molehr/gaaa084 ◽

2021 ◽

Author(s):

Chu-Chun Huang ◽

Chia-Hung Chou ◽

Yu-Shih Yang ◽

Hong-Nerng Ho ◽

Chia-Tung Shun ◽

...

Keyword(s):

Ovarian Function ◽

Primordial Follicle ◽

Inhibitory Effect ◽

Metformin Treatment ◽

High Power Field ◽

Primordial Follicles ◽

Apoptotic Effect ◽

Apoptotic Effects

Abstract Cyclophosphamide (CP) could cause severe gonadotoxicity via imbalanced activation of primordial follicles through PI3K/AKT/mTOR activation. Whether metformin, a widely prescribed anti-diabetes agent with mTOR inhibitory effect, could preserve ovarian function against CP toxicity is unknown. Female C57BL/6 mice were randomized into seven groups (n = 11), including control, CP-alone, CP+metformin, CP+sirolimus or everolimus, metformin-alone and sirolimus-alone groups. The duration of pharmaceutical treatment was four weeks. CP treatment significantly impaired ovarian function and fertility in mice. CP+metformin treatment significantly attenuated the gonadotoxicity comparing to CP-alone treatment (Primordial follicle count: 17.6 ± 4.2 versus 10.3 ± 2.7 follicles/high-power field; p = 0.027). CP+metformin treatment also tended to increase antral follicular count (5.4 ± 1.1 versus 2.5 ± 1.6 follicles/section), serum AMH levels (4.6 ± 1.2 versus 2.0 ± 0.8 ng/ml) and the litter size (4.2 ± 1.3 versus 1.5 ± 1.0 mice per pregnancy), compared with CP-alone group. Expression of phospho-mTOR and the number of TUNEL-positive granulosa cells increased after CP treatment and decreased in the CP+metformin groups, suggesting the mTOR inhibitory and anti-apoptotic effects of metformin. In in vitro granulosa cell experiments, the anti-apoptotic effect of metformin was blocked after inhibiting p53 or p21 function, and the expression of p53 mRNA was blocked with AMPK inhibitor, suggesting that the anti-apoptotic effect was AMPK/p53/p21-mediated. In conclusion, concurrent metformin treatment during CP therapy could significantly preserve ovarian function and fertility and could be a promising novel fertility preserving agent during chemotherapy. The relatively acceptable cost and well-established long-term safety profiles of this old drug might prompt its further clinical application at a faster pace.

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The mouse Balbiani body maintains primordial follicle quiescence via RNA storage

10.1101/2020.01.17.911040 ◽

2020 ◽

Author(s):

Lei Lei ◽

Kanako Ikami ◽

Haley Abbott ◽

Shiying Jin

Keyword(s):

Ovarian Function ◽

Follicular Development ◽

Primordial Follicle ◽

Primordial Follicles ◽

Mrna Decapping ◽

Balbiani Body ◽

Mrna Capping ◽

Capping Enzyme ◽

Decapping Enzyme

AbstractIn mammalian females, the transition between quiescent primordial follicles and follicular development is critical for maintaining ovarian function and reproductive longevity. In primary oocytes of mouse quiescent primordial follicles, Golgi complexes are organized into a spherical structure, the Balbiani body. Here, we show that the structure of the B-body is maintained by microtubules and actin. The B-body stores mRNA-capping enzyme and 597 mRNAs associated with mRNA-decapping enzyme 1A. Proteins encoded by these mRNAs function in enzyme binding, cellular component organization and packing of telomere ends. Pharmacological disassembly of the B-body triggers translation of stored mRNAs and activates primordial follicles in culture and in vivo mouse model. Thus, primordial follicle quiescence is maintained by the B-body, and translationally inactive B-body-stored mRNAs may be regulated by 5’-capping.

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Cisplatin- and cyclophosphamide-induced primordial follicle depletion is caused by direct damage to oocytes

MHR Basic science of reproductive medicine ◽

10.1093/molehr/gaz020 ◽

2019 ◽

Vol 25 (8) ◽

pp. 433-444 ◽

Cited By ~ 23

Author(s):

Q N Nguyen ◽

N Zerafa ◽

S H Liew ◽

J K Findlay ◽

M Hickey ◽

...

Keyword(s):

Ovarian Reserve ◽

Molecular Mechanisms ◽

Ovarian Function ◽

Primordial Follicle ◽

Cell Types ◽

Tunel Staining ◽

Specific Cell ◽

Primordial Follicles ◽

Cellular Targets ◽

Direct Damage

Abstract It is well established that DNA-damaging chemotherapies can cause infertility and ovarian endocrine failure by depleting the ovarian reserve of primordial follicles. Currently, no effective pharmacological therapies exist for the preservation of long-term fertility and ovarian function in female cancer patients, due to a limited understanding of the mechanisms of chemotherapy-induced follicle depletion. This study investigated the cellular targets, molecular mechanisms, and temporal course of ovarian reserve depletion following treatment with commonly used chemotherapeutic drugs. Adult female C57BL/6 mice were injected i.p. with saline, cisplatin (5mg/kg), or cyclophosphamide (300mg/kg); ovaries were harvested after 8 or 24 hours. Follicle counts showed depletion of all follicular stages 24 hours after administration of cisplatin or cyclophosphamide. Eight hours post-treatment, H2A histone family member X (γH2AX) immunofluorescence showed DNA double-stranded breaks at all follicular stages, including within primordial follicle oocytes. This staining was resolving by 24 hours, indicating that primordial follicle oocytes begin to undergo either apoptosis or repair in this timeframe. γH2AX-positive follicles were further examined to identify the specific cell types damaged. In primordial, transitional, and primary follicles, only oocytes sustained DNA damage, whereas in secondary and antral follicles, only somatic cells were affected. TUNEL staining confirmed that apoptosis occurs in these targeted cell types. Whilst multi-drug and multi-dose regimens were not examined, this study conclusively shows that cyclophosphamide and cisplatin cause direct damage to primordial follicle oocytes, which then undergo apoptosis. Therefore, future pharmacological strategies to prevent chemotherapy-induced infertility in females must specifically prevent primordial follicle oocyte death.

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Hormonal suppression for fertility preservation in males and females

Reproduction ◽

10.1530/rep-08-0096 ◽

2008 ◽

Vol 136 (6) ◽

pp. 691-701 ◽

Cited By ~ 60

Author(s):

Marvin L Meistrich ◽

Gunapala Shetty

Keyword(s):

Clinical Trials ◽

Randomized Clinical Trials ◽

Ovarian Function ◽

Follicular Development ◽

Primordial Follicle ◽

Cytotoxic Agents ◽

Cytotoxic Therapy ◽

Primordial Follicles ◽

Beneficial Effects ◽

Gnrh Analogs

Methods to restore fertility of men and women sterilized by medical treatments and environmental toxicant exposures are under investigation. Rendering spermatogenesis and ovarian follicular development kinetically quiescent by suppression of gonadotropins has been proposed to protect them from damage by cytotoxic therapy. Although the method fails to protect the fertility of male mice and monkeys, gonadotropin and testosterone suppression in rats before or after cytotoxic therapy do enhance the recovery of spermatogenesis. However, the mechanism involves not the induction of quiescence but rather the reversal, by suppression of testosterone, of a block in differentiation of surviving spermatogonia caused by damage to the somatic environment. In men, only one of eight clinical trials was successful in protecting or restoring spermatogenesis after cytotoxic therapy. In women, protection of primordial follicles in several species from damage by cytotoxic agents using GnRH analogs has been claimed; however, only two studies in mice appear convincing. The protection cannot involve the induction of quiescence in the already dormant primordial follicle but may involve direct effects of GnRH analogs or indirect effects of gonadotropin suppression on the whole ovary. Although numerous studies in female patients undergoing chemotherapy indicate that GnRH analogs might be protective of ovarian function, none of the studies showing protection were prospective randomized clinical trials and thus they are inconclusive. Considering interspecies differences and similarities in the gonadal sensitivity to cytotoxic agents and hormones, mechanistic studies are needed to identify the specific beneficial effects of hormonal suppression in select animal models that may be applicable to humans.

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Altered germline cyst and oocyte differentiation in Tex14 mutant mice reveal a new mechanism underlying female reproductive life-span

10.1101/814384 ◽

2019 ◽

Author(s):

Nafisa Nuzhat ◽

Kanako Ikami ◽

Haley Abbott ◽

Heather Tanner ◽

Allan C. Spradling ◽

...

Keyword(s):

Germ Cells ◽

Ovarian Function ◽

Primordial Follicle ◽

Wild Type ◽

Developmental Potential ◽

Primordial Follicles ◽

Intercellular Bridges ◽

Reproductive Life ◽

Reproductive Life Span ◽

Oocyte Differentiation

AbstractIn adult mammalian females, primordial follicles that form in the fetal/neonatal ovary are the only source to sustain adult ovarian function. Our previous studies revealed that during oocyte differentiation and primordial follicle formation in mouse fetal ovaries, primary oocytes form via gaining cytoplasm and organelles from sister germ cells that are connected to them by intercellular bridges within germline cysts. To better understand the role of intercellular bridges in oocyte differentiation, we analyzed mutant females lacking testis-expressed 14 (Tex14), a gene involved in cytokinesis and bridge formation. In Tex14-/- fetal ovaries, germ cells divide to form a reduced number of cysts in which sister germ cells are still connected via syncytia or fragmented cell membranes, rather than normal intercellular bridges. Compared with wildtype cysts, Tex14-/- cysts fragment at a higher frequency and produce a greatly reduced number of primary oocytes with highly precocious cytoplasmic enrichment and enlarged volume. By contrast, Tex14+/- germline cysts are less fragmented and generate primary oocytes that are smaller than wild type. Interestingly, enlarged Tex14-/- primary oocytes are much more stable than wild type oocytes and more efficiently sustain folliculogenesis, whereas undersized Tex14+/- primary oocytes turn over at an accelerated rate. Our observations directly link the nature of fetal germ cell connectivity to cytoplasmic enrichment during oocyte differentiation and to oocyte developmental potential in the adult ovary. Our results imply that the duration of adult ovarian function is strongly influenced by the number of primary oocytes acquiring highly enriched cytoplasm during oocyte differentiation in fetal ovaries, rather than just by the size of the primordial follicle pool.

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Reproductive Longevity and Aging: Geroscience Approaches to Maintain Long-Term Ovarian Fitness

The Journals of Gerontology Series A ◽

10.1093/gerona/glaa204 ◽

2020 ◽

Author(s):

Natalia Llarena ◽

Christopher Hine

Keyword(s):

Ovarian Reserve ◽

Ovarian Function ◽

Reproductive Function ◽

Primordial Follicle ◽

Oocyte Quality ◽

Nutrient Sensing ◽

Primordial Follicles ◽

Age Related ◽

Reproductive Life ◽

Reproductive Life Span

Abstract Increases in delayed childbearing worldwide have elicited the need for a better understanding of the biological underpinnings and implications of age-related infertility. In women 35 years and older the incidences of infertility, aneuploidy, and birth defects dramatically increase. These outcomes are a result of age-related declines in both ovarian reserve and oocyte quality. In addition to waning reproductive function, the decline in estrogen secretion at menopause contributes to multisystem aging and the initiation of frailty. Both reproductive and hormonal ovarian function are limited by the primordial follicle pool, which is established in utero and declines irreversibly until menopause. Because ovarian function is dependent on the primordial follicle pool, an understanding of the mechanisms that regulate follicular growth and maintenance of the primordial follicle pool is critical for the development of interventions to prolong the reproductive life span. Multiple pathways related to aging and nutrient-sensing converge in the mammalian ovary to regulate quiescence or activation of primordial follicles. The PI3K/PTEN/AKT/FOXO3 and associated TSC/mTOR pathways are central to the regulation of the primordial follicle pool; however, aging-associated systems such as the insulin-like growth factor-1/growth hormone pathway, and transsulfuration/hydrogen sulfide pathways may also play a role. Additionally, sirtuins aid in maintaining developmental metabolic competence and chromosomal integrity of the oocyte. Here we review the pathways that regulate ovarian reserve and oocyte quality, and discuss geroscience interventions that leverage our understanding of these pathways to promote reproductive longevity.

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A novel marsupial pri-miRNA transcript has a putative role in gamete maintenance and defines a vertebrate miRNA cluster paralogous to the miR-15a/miR-16-1 cluster

Reproduction ◽

10.1530/rep-11-0208 ◽

2011 ◽

Vol 142 (4) ◽

pp. 539-550 ◽

Cited By ~ 2

Author(s):

Phil Chi Khang Au ◽

Stephen Frankenberg ◽

Lynne Selwood ◽

Mary Familari

Keyword(s):

Molecular Mechanisms ◽

Primordial Germ Cells ◽

Primordial Follicle ◽

Bioinformatic Analysis ◽

Male Reproduction ◽

Follicle Cells ◽

Mirna Cluster ◽

Supporting Cells ◽

Primordial Follicles ◽

Wide Range

Successful maintenance, survival and maturation of gametes rely on bidirectional communication between the gamete and its supporting cells. Before puberty, factors from the gamete and its supporting cells are necessary for spermatogonial stem cell and primordial follicle oocyte maintenance. Following gametogenesis, gametes rely on factors and nutrients secreted by cells of the reproductive tracts, the epididymis and/or oviduct, to complete maturation. Despite extensive studies on female and male reproduction, many of the molecular mechanisms of germ cell maintenance remain relatively unknown, particularly in marsupial species. We present the first study and characterisation of a novel primary miRNA transcript, pri-miR-16c, in the marsupial, the stripe-faced dunnart. Bioinformatic analysis showed that its predicted processed miRNA – miR-16c – is present in a wide range of vertebrates, but not eutherians. In situ hybridisation revealed dunnart pri-miR-16c expression in day 4 (primordial germ cells) and day 7 (oogonia) pouch young, in primary oocytes and follicle cells of primordial follicles but then only in follicle cells of primary, secondary and antral follicles in adult ovaries. In the adult testis, pri-miR-16c transcripts were present in the cytoplasm of spermatogonial cells. The oviduct and the epididymis both showed expression, but not any other somatic tissues examined or conceptuses during early embryonic development. This pattern of expression suggests that pri-miR-16c function may be associated with gamete maintenance, possibly through mechanisms involving RNA transfer, until the zygote enters the uterus at the pronuclear stage.

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