Publications by authors named "Eva R Hoffmann"

33 Publications

Characterization and Survival of Human Infant Testicular Cells After Direct Xenotransplantation.

Front Endocrinol (Lausanne) 2022 10;13:853482. Epub 2022 Mar 10.

Laboratory of Reproductive Biology, University Hospital of Copenhagen, Rigshospitalet, Copenhagen, Denmark.

Background: Cryopreservation of prepubertal testicular tissue preserves spermatogonial stem cells (SSCs) that may be used to restore fertility in men at risk of infertility due to gonadotoxic treatments for either a malignant or non-malignant disease. Spermatogonial stem cell-based transplantation is a promising fertility restoration technique. Previously, we performed xenotransplantation of propagated SSCs from prepubertal testis and found human SSCs colonies within the recipient testes six weeks post-transplantation. In order to avoid the propagation step of SSCs that may cause genetic and epigenetic changes, we performed direct injection of single cell suspension in this study, which potentially may be safer and easier to be applied in future clinical applications.

Methods: Testis biopsies were obtained from 11 infant boys (median age 1.3 years, range 0.5-3.5) with cryptorchidism. Following enzymatic digestion, dissociated single-cell suspensions were prelabeled with green fluorescent dye and directly transplanted into seminiferous tubules of busulfan-treated mice. Six to nine weeks post-transplantation, the presence of gonocytes and SSCs was determined by whole-mount immunofluorescence for a number of germ cell markers (MAGEA, GAGE, UCHL1, SALL4, UTF1, and LIN28), somatic cell markers (SOX9, CYP17A1).

Results: Following xenotransplantation human infant germ cells, consisting of gonocytes and SSCs, were shown to settle on the basal membrane of the recipient seminiferous tubules and form SSC colonies with expression of MAGEA, GAGE, UCHL1, SALL4, UTF1, and LIN28. The colonization efficiency was approximately 6%. No human Sertoli cells were detected in the recipient mouse testes.

Conclusion: Xenotransplantation, without propagation, of testicular cell suspensions from infant boys with cryptorchidism resulted in colonization of mouse seminiferous tubules six to nine weeks post-transplantation. Spermatogonial stem cell-based transplantation could be a therapeutic treatment for infertility of prepubertal boys with cryptorchidism and boys diagnosed with cancer. However, more studies are required to investigate whether the low number of the transplanted SSC is sufficient to secure the presence of sperm in the ejaculate of those patients over time.
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http://dx.doi.org/10.3389/fendo.2022.853482DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8960121PMC
April 2022

First come, first served: Mammalian recombination is timed to replication.

Cell 2021 08;184(16):4112-4114

DNRF Center for Chromosome Stability, Department of Cellular and Molecular Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark. Electronic address:

Meiotic recombination drives the formation of new chromosomes in germ cells and is essential for fertility in mammals. In this issue of Cell, Pratto et al. have developed a method to map replication origins directly in mammalian tissue for the first time, revealing evolutionary conservation between replication timing and meiotic recombination in males.
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http://dx.doi.org/10.1016/j.cell.2021.07.014DOI Listing
August 2021

Genetic insights into biological mechanisms governing human ovarian ageing.

Nature 2021 08 4;596(7872):393-397. Epub 2021 Aug 4.

Genome Integrity and Instability Group, Institut de Biotecnologia i Biomedicina, Universitat Autònoma de Barcelona, Cerdanyola del Vallès, Spain.

Reproductive longevity is essential for fertility and influences healthy ageing in women, but insights into its underlying biological mechanisms and treatments to preserve it are limited. Here we identify 290 genetic determinants of ovarian ageing, assessed using normal variation in age at natural menopause (ANM) in about 200,000 women of European ancestry. These common alleles were associated with clinical extremes of ANM; women in the top 1% of genetic susceptibility have an equivalent risk of premature ovarian insufficiency to those carrying monogenic FMR1 premutations. The identified loci implicate a broad range of DNA damage response (DDR) processes and include loss-of-function variants in key DDR-associated genes. Integration with experimental models demonstrates that these DDR processes act across the life-course to shape the ovarian reserve and its rate of depletion. Furthermore, we demonstrate that experimental manipulation of DDR pathways highlighted by human genetics increases fertility and extends reproductive life in mice. Causal inference analyses using the identified genetic variants indicate that extending reproductive life in women improves bone health and reduces risk of type 2 diabetes, but increases the risk of hormone-sensitive cancers. These findings provide insight into the mechanisms that govern ovarian ageing, when they act, and how they might be targeted by therapeutic approaches to extend fertility and prevent disease.
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http://dx.doi.org/10.1038/s41586-021-03779-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7611832PMC
August 2021

Origins and mechanisms leading to aneuploidy in human eggs.

Prenat Diagn 2021 04 22;41(5):620-630. Epub 2021 Mar 22.

Department of Genetics, Rutgers, The State University of New Jersey, Piscataway, New Jersey, USA.

The gain or loss of a chromosome-or aneuploidy-acts as one of the major triggers for infertility and pregnancy loss in humans. These chromosomal abnormalities affect more than 40% of eggs in women at both ends of the age spectrum, that is, young girls as well as women of advancing maternal age. Recent studies in human oocytes and embryos using genomics, cytogenetics, and in silico modeling all provide new insight into the rates and potential genetic and cellular factors associated with aneuploidy at varying stages of development. Here, we review recent studies that are shedding light on potential molecular mechanisms of chromosome missegregation in oocytes and embryos across the entire female reproductive life span.
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http://dx.doi.org/10.1002/pd.5927DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8237340PMC
April 2021

Chromosomal mosaicism: Origins and clinical implications in preimplantation and prenatal diagnosis.

Prenat Diagn 2021 04 22;41(5):631-641. Epub 2021 Mar 22.

Research and Development, Cytogenetics and Medical Genetics Unit, TOMA Advanced Biomedical Assays, S.p.A. (Impact Lab), Busto Arsizio, Varese, Italy.

The diagnosis of chromosomal mosaicism in the preimplantation and prenatal stage is fraught with uncertainty and multiple factors need to be considered in order to gauge the likely impact. The clinical effects of chromosomal mosaicism are directly linked to the type of the imbalance (size, gene content, and copy number), the timing of the initial event leading to mosaicism during embryogenesis/fetal development, the distribution of the abnormal cells throughout the various tissues within the body as well as the ratio of normal/abnormal cells within each of those tissues. Additional factors such as assay noise and culture artifacts also have an impact on the significance and management of mosaic cases. Genetic counseling is an important part of educating patients about the likelihood of having a liveborn with a chromosome abnormality and these risks differ according to the time of ascertainment and the tissue where the mosaic cells were initially discovered. Each situation needs to be assessed on a case-by-case basis and counseled accordingly. This review will discuss the clinical impact of finding mosaicism through: embryo biopsy, chorionic villus sampling, amniocentesis, and noninvasive prenatal testing using cell-free DNA.
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http://dx.doi.org/10.1002/pd.5931DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8176867PMC
April 2021

Genome diversity and instability in human germ cells and preimplantation embryos.

Semin Cell Dev Biol 2021 05 23;113:132-147. Epub 2021 Jan 23.

DNRF Center for Chromosome Stability, Department of Cellular and Molecular Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark. Electronic address:

Genome diversity is essential for evolution and is of fundamental importance to human health. Generating genome diversity requires phases of DNA damage and repair that can cause genome instability. Humans have a high incidence of de novo congenital disorders compared to other organisms. Recent access to eggs, sperm and preimplantation embryos is revealing unprecedented rates of genome instability that may result in infertility and de novo mutations that cause genomic imbalance in at least 70% of conceptions. The error type and incidence of de novo mutations differ during developmental stages and are influenced by differences in male and female meiosis. In females, DNA repair is a critical factor that determines fertility and reproductive lifespan. In males, aberrant meiotic recombination causes infertility, embryonic failure and pregnancy loss. Evidence suggest germ cells are remarkably diverse in the type of genome instability that they display and the DNA damage responses they deploy. Additionally, the initial embryonic cell cycles are characterized by a high degree of genome instability that cause congenital disorders and may limit the use of CRISPR-Cas9 for heritable genome editing.
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http://dx.doi.org/10.1016/j.semcdb.2020.12.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8097364PMC
May 2021

Publisher Correction: Regulation of the MLH1-MLH3 endonuclease in meiosis.

Nature 2021 Feb;590(7844):E29

Institute for Research in Biomedicine, Faculty of Biomedical Sciences, Università della Svizzera italiana (USI), Bellinzona, Switzerland.

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http://dx.doi.org/10.1038/s41586-020-03111-9DOI Listing
February 2021

Cas9 in Human Embryos: On Target but No Repair.

Cell 2020 12;183(6):1464-1466

Unitat de Citologia i Histologia, Departament de Biologia Cel·lular, Fisiologia i Immunologia, Facultat de Biociències, Universitat Autònoma de Barcelona, Cerdanyola del Vallès, Spain; Grup d'Inestabilitat i Integritat del Genoma, Institut de Biotecnologia i Biomedicina, Universitat Autònoma de Barcelona, Cerdanyola del Vallès, Spain.

In this issue of Cell, Zuccaro and colleagues show that on-target Cas9-mediated double-strand breaks cause chromosome loss or mis-repair of the disease allele in > 90% of human embryos. End joining repair pathways dominate, causing small insertions or deletions, which raises serious questions about using double-strand breaks for "gene surgery".
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http://dx.doi.org/10.1016/j.cell.2020.11.022DOI Listing
December 2020

Identification of a unique epigenetic profile in women with diminished ovarian reserve.

Fertil Steril 2021 03 4;115(3):732-741. Epub 2020 Dec 4.

Department of Obstetrics and Gynaecology, Department of Reproductive Medicine, Hospital Herlev, Copenhagen University, Copenhagen, Denmark.

Objective: To investigate whether epigenetic profiles of mural granulosa cells (MGC) and leukocytes from women with diminished ovarian reserve (DOR) differ from those of women with normal or high ovarian reserve.

Design: Prospectively collected material from a multicenter cohort of women undergoing fertility treatment.

Setting: Private and university-based facilities for clinical services and research.

Patient(s): One hundred and nineteen women of various ages and ovarian reserve status (antimüllerian hormone level) who provided blood samples and MGC.

Intervention(s): None.

Main Outcome Measure(s): Measures of epigenetic aging rates from whole-genome methylation array data: DNA methylation variability, age acceleration, DNA methylation telomere length estimator (DNAmTL), and accumulation of epimutations.

Result(s): Comparison of DOR or high ovarian reserve samples to controls (normal ovarian reserve) showed differential methylation variability between DOR and normal samples at 4,199 CpGs in MGC, and 447 between high and normal (false-discovery rate < 0.05). Variable sites in MGC from DOR were enriched in regions marked with the repressive histone modification H3K27me3, and also included genes involved in folliculogenesis, such as insulin growth factor 2 (IGF2) and antimüllerian hormone (AMH). Regardless of ovarian reserve, very few signals were detected in leukocytes, and no overlaps with those in MGC were found. Furthermore, we found a higher number of epimutations in MGC from women with DOR (Kruskal-Wallis test, difference in mean = 3,485).

Conclusion(s): The somatic cells of human ovarian follicles have a distinctive epigenetic profile in women with DOR. A high frequency of epimutations suggests premature aging. Ovarian reserve status was not reflected in the leukocyte epigenetic profile.
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http://dx.doi.org/10.1016/j.fertnstert.2020.09.009DOI Listing
March 2021

Preconception genome medicine: current state and future perspectives to improve infertility diagnosis and reproductive and health outcomes based on individual genomic data.

Hum Reprod Update 2021 02;27(2):254-279

Igenomix Foundation, INCLIVA, Valencia, Spain.

Background: Our genetic code is now readable, writable and hackable. The recent escalation of genome-wide sequencing (GS) applications in population diagnostics will not only enable the assessment of risks of transmitting well-defined monogenic disorders at preconceptional stages (i.e. carrier screening), but also facilitate identification of multifactorial genetic predispositions to sub-lethal pathologies, including those affecting reproductive fitness. Through GS, the acquisition and curation of reproductive-related findings will warrant the expansion of genetic assessment to new areas of genomic prediction of reproductive phenotypes, pharmacogenomics and molecular embryology, further boosting our knowledge and therapeutic tools for treating infertility and improving women's health.

Objective And Rationale: In this article, we review current knowledge and potential development of preconception genome analysis aimed at detecting reproductive and individual health risks (recessive genetic disease and medically actionable secondary findings) as well as anticipating specific reproductive outcomes, particularly in the context of IVF. The extension of reproductive genetic risk assessment to the general population and IVF couples will lead to the identification of couples who carry recessive mutations, as well as sub-lethal conditions prior to conception. This approach will provide increased reproductive autonomy to couples, particularly in those cases where preimplantation genetic testing is an available option to avoid the transmission of undesirable conditions. In addition, GS on prospective infertility patients will enable genome-wide association studies specific for infertility phenotypes such as predisposition to premature ovarian failure, increased risk of aneuploidies, complete oocyte immaturity or blastocyst development failure, thus empowering the development of true reproductive precision medicine.

Search Methods: Searches of the literature on PubMed Central included combinations of the following MeSH terms: human, genetics, genomics, variants, male, female, fertility, next generation sequencing, genome exome sequencing, expanded carrier screening, secondary findings, pharmacogenomics, controlled ovarian stimulation, preconception, genetics, genome-wide association studies, GWAS.

Outcomes: Through PubMed Central queries, we identified a total of 1409 articles. The full list of articles was assessed for date of publication, limiting the search to studies published within the last 15 years (2004 onwards due to escalating research output of next-generation sequencing studies from that date). The remaining articles' titles were assessed for pertinence to the topic, leaving a total of 644 articles. The use of preconception GS has the potential to identify inheritable genetic conditions concealed in the genome of around 4% of couples looking to conceive. Genomic information during reproductive age will also be useful to anticipate late-onset medically actionable conditions with strong genetic background in around 2-4% of all individuals. Genetic variants correlated with differential response to pharmaceutical treatment in IVF, and clear genotype-phenotype associations are found for aberrant sperm types, oocyte maturation, fertilization or pre- and post-implantation embryonic development. All currently known capabilities of GS at the preconception stage are reviewed along with persisting and forthcoming barriers for the implementation of precise reproductive medicine.

Wider Implications: The expansion of sequencing analysis to additional monogenic and polygenic traits may enable the development of cost-effective preconception tests capable of identifying underlying genetic causes of infertility, which have been defined as 'unexplained' until now, thus leading to the development of a true personalized genomic medicine framework in reproductive health.
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http://dx.doi.org/10.1093/humupd/dmaa044DOI Listing
February 2021

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Antibodies at Delivery in Women, Partners, and Newborns.

Obstet Gynecol 2021 01;137(1):49-55

Department of Obstetrics and Gynaecology, Copenhagen University Hospital Hvidovre, Hvidovre, the Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Copenhagen, Methods and Analysis, Statistics Denmark, Copenhagen, the Recurrent Pregnancy Loss Unit, the Capital Region, Rigshospitalet, Copenhagen University Hospital, Copenhagen, the Department of Clinical Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, the Department of Obstetrics and Gynaecology, the Fertility Clinic, Copenhagen University Hospital Hvidovre, Hvidovre, the Department of Clinical Microbiology, Copenhagen University Hospital Hvidovre, Hvidovre, the DNRF Center for Chromosome Stability (CCS), Department of Cellular and Molecular Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, the Fetal Medicine Unit, Department of Obstetrics and Gynaecology, Copenhagen University Hospital Hvidovre, Hvidovre, and the Department of Clinical Biochemistry, Copenhagen University Hospital Hvidovre, Hvidovre, Denmark.

Objective: To investigate the frequency of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies in parturient women, their partners, and their newborns and the association of such antibodies with obstetric and neonatal outcomes.

Methods: From April 4 to July 3, 2020, in a single university hospital in Denmark, all parturient women and their partners were invited to participate in the study, along with their newborns. Participating women and partners had a pharyngeal swab and a blood sample taken at admission; immediately after delivery, a blood sample was drawn from the umbilical cord. The swabs were analyzed for SARS-CoV-2 RNA by polymerase chain reaction, and the blood samples were analyzed for SARS-CoV-2 antibodies. Full medical history and obstetric and neonatal information were available.

Results: A total of 1,313 parturient women (72.5.% of all women admitted for delivery at the hospital in the study period), 1,188 partners, and 1,206 newborns participated in the study. The adjusted serologic prevalence was 2.6% in women and 3.5% in partners. Seventeen newborns had SARS-CoV-2 immunoglobulin G (IgG) antibodies, and none had immunoglobulin M antibodies. No associations between SARS-CoV-2 antibodies and obstetric or neonatal complications were found (eg, preterm birth, preeclampsia, cesarean delivery, Apgar score, low birth weight, umbilical arterial pH, need for continuous positive airway pressure, or neonatal admission), but statistical power to detect such differences was low. Full serologic data from 1,051 families showed an absolute risk of maternal infection of 39% if the partner had antibodies.

Conclusion: We found no association between SARS-CoV-2 infection and obstetric or neonatal complications. Sixty-seven percent of newborns delivered by mothers with antibodies had SARS-CoV-2 IgG antibodies. A limitation of our study is that we lacked statistical power to detect small but potentially meaningful differences between those with and without evidence of infection.
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http://dx.doi.org/10.1097/AOG.0000000000004199DOI Listing
January 2021

Parental Acceptance Rate of Testicular Tissue Cryopreservation in Danish Boys with Cryptorchidism.

Sex Dev 2019 20;13(5-6):246-257. Epub 2020 Oct 20.

Despite orchidopexy within the first year of life, 20-25% of boys with nonsyndromic cryptorchidism may risk infertility according to histological and hormonal data obtained during surgery. The aim of this study was to evaluate the acceptance rate of testicular tissue cryopreservation among parents of prepubertal boys with cryptorchidism. Fourteen boys with cryptorchidism and high infertility risk were offered cryopreservation as an additional procedure after orchidopexy based on abnormal histopathological findings at primary surgery, whereas 27 boys with bilateral cryptorchidism were offered cryopreservation at the initial orchidopexy. A total of 90% of parents (37/41, 13/14, and 24/27) gave consent to perform cryopreservation, despite being well-informed that the procedural efficacy is largely unproven and may only be needed in about 20% of cases. The number of germ cells per tubule cross-section was 0.03-1.70 (median 0.37) and 22 boys (54%, 22/41) had a value below the lower range. Twelve boys (29%, 12/41) had no type A dark spermatogonia in their biopsy. Cryopreservation of testicular tissue is the first step to introduce spermatogonial stem cell-based therapy into clinical male infertility treatment. At the time of orchidopexy, a testicular biopsy can be collected to ascertain the infertility risk, and it may be an option for boys with bilateral cryptorchidism to have spermatogonial stem cells frozen as a fertility reserve.
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http://dx.doi.org/10.1159/000511158DOI Listing
October 2020

Regulation of the MLH1-MLH3 endonuclease in meiosis.

Nature 2020 10 19;586(7830):618-622. Epub 2020 Aug 19.

Institute for Research in Biomedicine, Faculty of Biomedical Sciences, Università della Svizzera italiana (USI), Bellinzona, Switzerland.

During prophase of the first meiotic division, cells deliberately break their DNA. These DNA breaks are repaired by homologous recombination, which facilitates proper chromosome segregation and enables the reciprocal exchange of DNA segments between homologous chromosomes. A pathway that depends on the MLH1-MLH3 (MutLγ) nuclease has been implicated in the biased processing of meiotic recombination intermediates into crossovers by an unknown mechanism. Here we have biochemically reconstituted key elements of this pro-crossover pathway. We show that human MSH4-MSH5 (MutSγ), which supports crossing over, binds branched recombination intermediates and associates with MutLγ, stabilizing the ensemble at joint molecule structures and adjacent double-stranded DNA. MutSγ directly stimulates DNA cleavage by the MutLγ endonuclease. MutLγ activity is further stimulated by EXO1, but only when MutSγ is present. Replication factor C (RFC) and the proliferating cell nuclear antigen (PCNA) are additional components of the nuclease ensemble, thereby triggering crossing-over. Saccharomyces cerevisiae strains in which MutLγ cannot interact with PCNA present defects in forming crossovers. Finally, the MutLγ-MutSγ-EXO1-RFC-PCNA nuclease ensemble preferentially cleaves DNA with Holliday junctions, but shows no canonical resolvase activity. Instead, it probably processes meiotic recombination intermediates by nicking double-stranded DNA adjacent to the junction points. As DNA nicking by MutLγ depends on its co-factors, the asymmetric distribution of MutSγ and RFC-PCNA on meiotic recombination intermediates may drive biased DNA cleavage. This mode of MutLγ nuclease activation might explain crossover-specific processing of Holliday junctions or their precursors in meiotic chromosomes.
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http://dx.doi.org/10.1038/s41586-020-2592-2DOI Listing
October 2020

KDM4A regulates the maternal-to-zygotic transition by protecting broad H3K4me3 domains from H3K9me3 invasion in oocytes.

Nat Cell Biol 2020 04 30;22(4):380-388. Epub 2020 Mar 30.

DNRF Center for Chromosome Stability (CCS), Department of Cellular and Molecular Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.

The importance of germline-inherited post-translational histone modifications on priming early mammalian development is just emerging. Histone H3 lysine 9 (H3K9) trimethylation is associated with heterochromatin and gene repression during cell-fate change, whereas histone H3 lysine 4 (H3K4) trimethylation marks active gene promoters. Mature oocytes are transcriptionally quiescent and possess remarkably broad domains of H3K4me3 (bdH3K4me3). It is unknown which factors contribute to the maintenance of the bdH3K4me3 landscape. Lysine-specific demethylase 4A (KDM4A) demethylates H3K9me3 at promoters marked by H3K4me3 in actively transcribing somatic cells. Here, we report that KDM4A-mediated H3K9me3 demethylation at bdH3K4me3 in oocytes is crucial for normal pre-implantation development and zygotic genome activation after fertilization. The loss of KDM4A in oocytes causes aberrant H3K9me3 spreading over bdH3K4me3, resulting in insufficient transcriptional activation of genes, endogenous retroviral elements and chimeric transcripts initiated from long terminal repeats during zygotic genome activation. The catalytic activity of KDM4A is essential for normal epigenetic reprogramming and pre-implantation development. Hence, KDM4A plays a crucial role in preserving the maternal epigenome integrity required for proper zygotic genome activation and transfer of developmental control to the embryo.
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http://dx.doi.org/10.1038/s41556-020-0494-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7212036PMC
April 2020

Review of injection techniques for spermatogonial stem cell transplantation.

Hum Reprod Update 2020 04;26(3):368-391

Biology of the Testis, Research Laboratory for Reproduction, Genetics and Regenerative Medicine, Vrije Universiteit Brussel (VUB), 1090 Brussels, Belgium.

Background: Although the prognosis of childhood cancer survivors has increased dramatically during recent years, chemotherapy and radiation treatments for cancer and other conditions may lead to permanent infertility in prepubertal boys. Recent developments have shown that spermatogonial stem cell (SSC) transplantation may be a hope for restoring fertility in adult survivors of childhood cancers. For this reason, several centres around the world are collecting and cryopreserving testicular tissue or cells anticipating that, in the near future, some patients will return for SSC transplantation. This review summarizes the current knowledge and utility of SSC transplantation techniques.

Objective And Rationale: The aim of this narrative review is to provide an overview of the currently used experimental injection techniques for SSC transplantation in animal and human testes. This is crucial in understanding and determining the role of the different techniques necessary for successful transplantation.

Search Methods: A comprehensive review of peer-reviewed publications on this topic was performed using the PubMed and Google Scholar databases. The search was limited to English language work and studies between 1994 (from the first study on SSC transplantation) and April 2019. Key search terms included mouse, rat, boar, ram, dog, sheep, goat, cattle, monkey, human, cadaver, testes, SSC transplantation, injection and technique.

Outcomes: This review provides an extensive clinical overview of the current research in the field of human SSC transplantation. Rete testis injection with ultrasonography guidance currently seems the most promising injection technique thus far; however, the ability to draw clear conclusions is limited due to long ischemia time of cadaver testis, the relatively decreased volume of the testis, the diminishing size of seminiferous tubules, a lack of intratesticular pressure and leakage into the interstitium during the injection on human cadaver testis. Current evidence does not support improved outcomes from multiple infusions through the rete testes. Overall, further optimization is required to increase the efficiency and safety of the infusion method.

Wider Implications: Identifying a favourable injection method for SSC transplantation will provide insight into the mechanisms of successful assisted human reproduction. Future research could focus on reducing leakage and establishing the optimal infusion cell concentrations and pressure.
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http://dx.doi.org/10.1093/humupd/dmaa003DOI Listing
April 2020

Genetic predisposition to mosaic Y chromosome loss in blood.

Nature 2019 11 20;575(7784):652-657. Epub 2019 Nov 20.

Genetics of Complex Traits, University of Exeter Medical School, University of Exeter, Exeter, UK.

Mosaic loss of chromosome Y (LOY) in circulating white blood cells is the most common form of clonal mosaicism, yet our knowledge of the causes and consequences of this is limited. Here, using a computational approach, we estimate that 20% of the male population represented in the UK Biobank study (n = 205,011) has detectable LOY. We identify 156 autosomal genetic determinants of LOY, which we replicate in 757,114 men of European and Japanese ancestry. These loci highlight genes that are involved in cell-cycle regulation and cancer susceptibility, as well as somatic drivers of tumour growth and targets of cancer therapy. We demonstrate that genetic susceptibility to LOY is associated with non-haematological effects on health in both men and women, which supports the hypothesis that clonal haematopoiesis is a biomarker of genomic instability in other tissues. Single-cell RNA sequencing identifies dysregulated expression of autosomal genes in leukocytes with LOY and provides insights into why clonal expansion of these cells may occur. Collectively, these data highlight the value of studying clonal mosaicism to uncover fundamental mechanisms that underlie cancer and other ageing-related diseases.
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http://dx.doi.org/10.1038/s41586-019-1765-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6887549PMC
November 2019

Computational and cellular studies reveal structural destabilization and degradation of MLH1 variants in Lynch syndrome.

Elife 2019 11 7;8. Epub 2019 Nov 7.

Department of Biology, The Linderstrøm-Lang Centre for Protein Science, University of Copenhagen, Copenhagen, Denmark.

Defective mismatch repair leads to increased mutation rates, and germline loss-of-function variants in the repair component MLH1 cause the hereditary cancer predisposition disorder known as Lynch syndrome. Early diagnosis is important, but complicated by many variants being of unknown significance. Here we show that a majority of the disease-linked MLH1 variants we studied are present at reduced cellular levels. We show that destabilized MLH1 variants are targeted for chaperone-assisted proteasomal degradation, resulting also in degradation of co-factors PMS1 and PMS2. In silico saturation mutagenesis and computational predictions of thermodynamic stability of MLH1 missense variants revealed a correlation between structural destabilization, reduced steady-state levels and loss-of-function. Thus, we suggest that loss of stability and cellular degradation is an important mechanism underlying many variants in Lynch syndrome. Combined with analyses of conservation, the thermodynamic stability predictions separate disease-linked from benign variants, and therefore hold potential for Lynch syndrome diagnostics.
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http://dx.doi.org/10.7554/eLife.49138DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6837844PMC
November 2019

Meiotic Kinetochores Fragment into Multiple Lobes upon Cohesin Loss in Aging Eggs.

Curr Biol 2019 11 31;29(22):3749-3765.e7. Epub 2019 Oct 31.

Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, Göttingen 37077, Germany. Electronic address:

Chromosome segregation errors during female meiosis are a leading cause of pregnancy loss and human infertility. The segregation of chromosomes is driven by interactions between spindle microtubules and kinetochores. Kinetochores in mammalian oocytes are subjected to special challenges: they need to withstand microtubule pulling forces over multiple hours and are built on centromeric chromatin that in humans is decades old. In meiosis I, sister kinetochores are paired and oriented toward the same spindle pole. It is well established that they progressively separate from each other with advancing female age. However, whether aging also affects the internal architecture of centromeres and kinetochores is currently unclear. Here, we used super-resolution microscopy to study meiotic centromere and kinetochore organization in metaphase-II-arrested eggs from three mammalian species, including humans. We found that centromeric chromatin decompacts with advancing maternal age. Kinetochores built on decompacted centromeres frequently lost their integrity and fragmented into multiple lobes. Fragmentation extended across inner and outer kinetochore regions and affected over 30% of metaphase-II-arrested (MII) kinetochores in aged women and mice, making the lobular architecture a prominent feature of the female meiotic kinetochore. We demonstrate that a partial cohesin loss, as is known to occur in oocytes with advancing maternal age, is sufficient to trigger centromere decompaction and kinetochore fragmentation. Microtubule pulling forces further enhanced the fragmentation and shaped the arrangement of kinetochore lobes. Fragmented kinetochores were frequently abnormally attached to spindle microtubules, suggesting that kinetochore fragmentation could contribute to the maternal age effect in mammalian eggs.
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http://dx.doi.org/10.1016/j.cub.2019.09.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6868511PMC
November 2019

Xeno-Free Propagation of Spermatogonial Stem Cells from Infant Boys.

Int J Mol Sci 2019 Oct 29;20(21). Epub 2019 Oct 29.

Laboratory of Reproductive Biology, Rigshospitalet, University Hospital of Copenhagen, 2100 Copenhagen, Denmark.

Spermatogonial stem cell (SSC) transplantation therapy is a promising strategy to renew spermatogenesis for prepubertal boys whose fertility is compromised. However, propagation of SSCs is required due to a limited number of SSCs in cryopreserved testicular tissue. This propagation must be done under xeno-free conditions for clinical application. SSCs were propagated from infant testicular tissue (7 mg and 10 mg) from two boys under xeno-free conditions using human platelet lysate and nutrient source. We verified SSC-like cell clusters (SSCLCs) by quantitative real-time polymerase chain reaction (PCR) and immune-reaction assay using the SSC markers undifferentiated embryonic cell transcription factor 1 (UTF1), ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL1), GDNF receptor alpha-1 (GFRα-1) Fα and promyelocytic leukaemia zinc finger protein (PLZF). The functionality of the propagated SSCs was investigated by pre-labelling using green fluorescent Cell Linker PKH67 and xeno-transplantation of the SSCLCs into busulfan-treated, therefore sterile, immunodeficient mice. SSC-like cell clusters (SSCLCs) appeared after 2 weeks in primary passage. The SSCLCs were SSC-like as the UTF1, UCHL1, GFRα1 and PLZF were all positive. After 2.5 months' culture period, a total of 13 million cells from one sample were harvested for xenotransplantation. Labelled human propagated SSCs were identified and verified in mouse seminiferous tubules at 3-6 weeks, confirming that the transplanted cells contain SSCLCs. The present xeno-free clinical culture protocol allows propagation of SSCs from infant boys.
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http://dx.doi.org/10.3390/ijms20215390DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6862004PMC
October 2019

Propagation of Spermatogonial Stem Cell-Like Cells From Infant Boys.

Front Physiol 2019 19;10:1155. Epub 2019 Sep 19.

Laboratory of Reproductive Biology, Copenhagen University Hospital, Copenhagen, Denmark.

Background: Gonadotoxic treatment of malignant diseases as well as some non-malignant conditions such as cryptorchidism in young boys may result in infertility and failure to father children later in life. As a fertility preserving strategy, several centers collect testicular biopsies to cryopreserve spermatogonial stem cells (SSCs) world-wide. One of the most promising therapeutic strategies is to transplant SSCs back into the seminiferous tubules to initiate endogenous spermatogenesis. However, to obtain sufficient numbers of SSC to warrant transplantation, propagation of cells is needed together with proper validation of their stem cell identity.

Materials And Methods: A minute amount of testicular biopsies (between 5 mg and 10 mg) were processed by mechanical and enzymatic digestion. SSCs were enriched by differential plating method in StemPro-34 medium supplemented with several growth factors. SSC-like cell clusters (SSCLCs) were passaged five times. SSCLCs were identified by immunohistochemical and immunofluorescence staining, using protein expression patterns in testis biopsies as reference. Quantitative polymerase chain reaction analysis of SSC markers LIN-28 homolog A (LIN28A), G antigen 1 (GAGE1), promyelocytic leukemia zinc finger protein (PLZF), integrin alpha 6 (ITGA6), ubiquitin carboxy-terminal hydrolase L1 (UCHL1) and integrin beta 1 (ITGB1) were also used to validate the SSC-like cell identity.

Results: Proliferation of SSCLCs was achieved. The presence of SSCs in SSCLCs was confirmed by positive immunostaining of LIN28, UCHL1 and quantitative polymerase chain reaction for LIN28A, UCHL1, PLZF, ITGA6, and ITGB1, respectively.

Conclusion: This study has demonstrated that SSCs from infant boys possess the capacity for proliferation and advance a fertility preservation strategy for pre-pubertal boys who may otherwise lose their fertility.
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http://dx.doi.org/10.3389/fphys.2019.01155DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6761273PMC
September 2019

Chromosome errors in human eggs shape natural fertility over reproductive life span.

Science 2019 09;365(6460):1466-1469

DNRF Center for Chromosome Stability, Department of Cellular and Molecular Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark.

Chromosome errors, or aneuploidy, affect an exceptionally high number of human conceptions, causing pregnancy loss and congenital disorders. Here, we have followed chromosome segregation in human oocytes from females aged 9 to 43 years and report that aneuploidy follows a U-curve. Specific segregation error types show different age dependencies, providing a quantitative explanation for the U-curve. Whole-chromosome nondisjunction events are preferentially associated with increased aneuploidy in young girls, whereas centromeric and more extensive cohesion loss limit fertility as women age. Our findings suggest that chromosomal errors originating in oocytes determine the curve of natural fertility in humans.
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http://dx.doi.org/10.1126/science.aav7321DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7212007PMC
September 2019

SureTypeSC-a Random Forest and Gaussian mixture predictor of high confidence genotypes in single-cell data.

Bioinformatics 2019 12;35(23):5055-5062

DNRF Center for Chromosome Stability, Department of Cellular and Molecular Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen N, Denmark.

Motivation: Accurate genotyping of DNA from a single cell is required for applications such as de novo mutation detection, linkage analysis and lineage tracing. However, achieving high precision genotyping in the single-cell environment is challenging due to the errors caused by whole-genome amplification. Two factors make genotyping from single cells using single nucleotide polymorphism (SNP) arrays challenging. The lack of a comprehensive single-cell dataset with a reference genotype and the absence of genotyping tools specifically designed to detect noise from the whole-genome amplification step. Algorithms designed for bulk DNA genotyping cause significant data loss when used for single-cell applications.

Results: In this study, we have created a resource of 28.7 million SNPs, typed at high confidence from whole-genome amplified DNA from single cells using the Illumina SNP bead array technology. The resource is generated from 104 single cells from two cell lines that are available from the Coriell repository. We used mother-father-proband (trio) information from multiple technical replicates of bulk DNA to establish a high quality reference genotype for the two cell lines on the SNP array. This enabled us to develop SureTypeSC-a two-stage machine learning algorithm that filters a substantial part of the noise, thereby retaining the majority of the high quality SNPs. SureTypeSC also provides a simple statistical output to show the confidence of a particular single-cell genotype using Bayesian statistics.

Availability And Implementation: The implementation of SureTypeSC in Python and sample data are available in the GitHub repository: https://github.com/puko818/SureTypeSC.

Supplementary Information: Supplementary data are available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/btz412DOI Listing
December 2019

In Vitro Maturation and Culture of Human Oocytes.

Methods Mol Biol 2018 ;1818:23-30

DNRF Center for Chromosome Stability, Department of Cellular and Molecular Medicine, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark.

We describe the collection, culture, and ex vivo, in vitro maturation of germinal vesicle (GV) oocytes obtained from human small antral follicles (hSAFs). hSAFs contain fully grown GV oocytes and have the advantages that they are more numerous than large or mature follicles, which are used in IVF treatment. hSAFs can be obtained directly from human ovarian tissue without exogenous gonadotrophin stimulation and therefore allows studies of oocytes even from young women and girls. The method described here was developed to study human female meiosis but could in theory also be used for fertility treatment.
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http://dx.doi.org/10.1007/978-1-4939-8603-3_3DOI Listing
March 2019

Single cell genomics to study DNA and chromosome changes in human gametes and embryos.

Methods Cell Biol 2018 24;144:441-457. Epub 2018 Apr 24.

Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Brighton, United Kingdom; Center for Chromosome Stability, University of Copenhagen, Copenhagen, Denmark. Electronic address:

Genomic and chromosomal changes occur with a high rate in the germline and preimplantation embryos. To study such changes directly in the germline of mammals requires access to material as well as single cell genomics. Recent improvements in embryology and single-cell DNA amplification make it possible to study the genomic changes directly in human oocytes, sperm, and preimplantation embryos. This is particularly important for the study of chromosome segregation directly in human oocytes and preimplantation embryos. Here, we present a practical approach how to obtain high quality DNA sequences and genotypes from single cells, using manual handling of the material that makes it possible to detect genomic changes in meiosis and mitosis spanning the entire range from single nucleotide changes to whole chromosome aneuploidies.
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http://dx.doi.org/10.1016/bs.mcb.2018.03.037DOI Listing
December 2018

Tripolar chromosome segregation drives the association between maternal genotype at variants spanning PLK4 and aneuploidy in human preimplantation embryos.

Hum Mol Genet 2018 07;27(14):2573-2585

School of Biosciences, University of Kent, Canterbury, UK.

Aneuploidy is prevalent in human embryos and is the leading cause of pregnancy loss. Many aneuploidies arise during oogenesis, increasing with maternal age. Superimposed on these meiotic aneuploidies are frequent errors occurring during early mitotic divisions, contributing to widespread chromosomal mosaicism. Here we reanalyzed a published dataset comprising preimplantation genetic testing for aneuploidy in 24 653 blastomere biopsies from day-3 cleavage-stage embryos, as well as 17 051 trophectoderm biopsies from day-5 blastocysts. We focused on complex abnormalities that affected multiple chromosomes simultaneously, seeking insights into their formation. In addition to well-described patterns such as triploidy and haploidy, we identified 4.7% of blastomeres possessing characteristic hypodiploid karyotypes. We inferred this signature to have arisen from tripolar chromosome segregation in normally fertilized diploid zygotes or their descendant diploid cells. This could occur via segregation on a tripolar mitotic spindle or by rapid sequential bipolar mitoses without an intervening S-phase. Both models are consistent with time-lapse data from an intersecting set of 77 cleavage-stage embryos, which were enriched for the tripolar signature among embryos exhibiting abnormal cleavage. The tripolar signature was strongly associated with common maternal genetic variants spanning the centrosomal regulator PLK4, driving the association we previously reported with overall mitotic errors. Our findings are consistent with the known capacity of PLK4 to induce tripolar mitosis or precocious M-phase upon dysregulation. Together, our data support tripolar chromosome segregation as a key mechanism generating complex aneuploidy in cleavage-stage embryos and implicate maternal genotype at a quantitative trait locus spanning PLK4 as a factor influencing its occurrence.
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http://dx.doi.org/10.1093/hmg/ddy147DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6030883PMC
July 2018

Human female meiosis revised: new insights into the mechanisms of chromosome segregation and aneuploidies from advanced genomics and time-lapse imaging.

Hum Reprod Update 2017 11;23(6):706-722

Clinica Valle Giulia, G.EN.E.R.A. Center for Reproductive Medicine, via G. De Notaris 2b, 00197 Rome, Italy.

Background: The unbalanced transmission of chromosomes in human gametes and early preimplantation embryos causes aneuploidy, which is a major cause of infertility and pregnancy failure. A baseline of 20% of human oocytes are estimated to be aneuploid and this increases exponentially from 30 to 35 years, reaching on average 80% by 42 years. As a result, reproductive senescence in human females is predominantly determined by the accelerated decline in genetic quality of oocytes from 30 years of age.

Objective And Rationale: Understanding mechanisms of chromosome segregation and aneuploidies in the female germline is a crucial step towards the development of new diagnostic approaches and, possibly, for the development of therapeutic targets and molecules. Here, we have reviewed emerging mechanisms that may drive human aneuploidy, in particular the maternal age effect.

Search Methods: We conducted a systematic search in PubMed Central of the primary literature from 1990 through 2016 following the PRISMA guidelines, using MeSH terms related to human aneuploidy. For model organism research, we conducted a literature review based on references in human oocytes manuscripts and general reviews related to chromosome segregation in meiosis and mitosis.

Outcomes: Advances in genomic and imaging technologies are allowing unprecedented insight into chromosome segregation in human oocytes. This includes the identification of a novel chromosome segregation error, termed reverse segregation, as well as sister kinetochore configurations that were not predicted based on murine models.

Wider Implications: Elucidation of mechanisms that result in errors in chromosome segregation in meiosis may lead to therapeutic developments that could improve reproductive outcomes by reducing aneuploidy.
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http://dx.doi.org/10.1093/humupd/dmx026DOI Listing
November 2017

Generation of meiomaps of genome-wide recombination and chromosome segregation in human oocytes.

Nat Protoc 2016 07 16;11(7):1229-43. Epub 2016 Jun 16.

The Bridge Centre, London, UK.

We have developed a protocol for the generation of genome-wide maps (meiomaps) of recombination and chromosome segregation for the three products of human female meiosis: the first and second polar bodies (PB1 and PB2) and the corresponding oocyte. PB1 is biopsied and the oocyte is artificially activated by exposure to calcium ionophore, after which PB2 is biopsied and collected with the corresponding oocyte. The whole genomes of the polar bodies and oocytes are amplified by multiple displacement amplification and, together with maternal genomic DNA, genotyped for ∼300,000 single-nucleotide polymorphisms (SNPs) genome-wide by microarray. Informative maternal heterozygous SNPs are phased using a haploid PB2 or oocyte as a reference. A simple algorithm is then used to identify the maternal haplotypes for each chromosome, in all of the products of meiosis for each oocyte. This allows mapping of crossovers and analysis of chromosome segregation patterns. The protocol takes a minimum of 3-5 d and requires a clinical embryologist with micromanipulation experience and a molecular biologist with basic bioinformatic skills. It has several advantages over previous methods; importantly, the use of artificial oocyte activation avoids the creation of embryos for research purposes. In addition, compared with next-generation sequencing, targeted SNP genotyping is cost-effective and it simplifies the bioinformatic analysis, as only one haploid reference sample is required to establish phase for maternal haplotyping. Finally, meiomapping is more informative than copy-number analysis alone for analysis of chromosome segregation patterns. Using this protocol, we have provided new insights that may lead to improvements in assisted reproduction for the treatment of infertility.
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http://dx.doi.org/10.1038/nprot.2016.075DOI Listing
July 2016

Genome-wide maps of recombination and chromosome segregation in human oocytes and embryos show selection for maternal recombination rates.

Nat Genet 2015 Jul 18;47(7):727-735. Epub 2015 May 18.

Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Brighton, UK.

Crossover recombination reshuffles genes and prevents errors in segregation that lead to extra or missing chromosomes (aneuploidy) in human eggs, a major cause of pregnancy failure and congenital disorders. Here we generate genome-wide maps of crossovers and chromosome segregation patterns by recovering all three products of single female meioses. Genotyping >4 million informative SNPs from 23 complete meioses allowed us to map 2,032 maternal and 1,342 paternal crossovers and to infer the segregation patterns of 529 chromosome pairs. We uncover a new reverse chromosome segregation pattern in which both homologs separate their sister chromatids at meiosis I; detect selection for higher recombination rates in the female germ line by the elimination of aneuploid embryos; and report chromosomal drive against non-recombinant chromatids at meiosis II. Collectively, our findings show that recombination not only affects homolog segregation at meiosis I but also the fate of sister chromatids at meiosis II.
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http://dx.doi.org/10.1038/ng.3306DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4770575PMC
July 2015

Distinct regulation of Mlh1p heterodimers in meiosis and mitosis in Saccharomyces cerevisiae.

Genetics 2010 Jun 9;185(2):459-67. Epub 2010 Apr 9.

Department of Genetics, University of Leicester, Leicester, LE1 7RH, United Kingdom.

Mlh1p forms three heterodimers that are important for mismatch repair (Mlh1p/Pms1p), crossing over during meiosis (Mlh1p/Mlh3p), and channeling crossover events into a specific pathway (Mlh1p/Mlh2p). All four proteins contain highly conserved ATPase domains and Pms1p has endonuclease activity. Studies of the functional requirements for Mlh1p/Pms1p in Saccharomyces cerevisae revealed an asymmetric contribution of the ATPase domains to repairing mismatches. Here we investigate the functional requirements of the Mlh1p and Mlh3p ATPase domains in meiosis by constructing separation of function mutations in Mlh3p. These mutations are analogous to mutations of Mlh1p that have been shown to lead to loss of ATP binding and/or ATP hydrolysis. Our data suggest that ATP binding by Mlh3p is required for meiotic crossing over while ATP hydrolysis is dispensable. This has been seen previously for Mlh1p. However, when mutations that affect ATP hydrolysis by both Mlh3p and Mlh1p are combined within a single cell, meiotic crossover frequencies are reduced. These observations suggest that the function of the Mlh1p/Mlh3p heterodimer requires both subunits to bind ATP but only one to efficiently hydrolyze it. Additionally, two different amino acid substitutions to the same residue (G97) in Mlh3p affect the minor mismatch repair function of Mlh3p while only one of them compromises its ability to promote crossing over. These studies thus reveal different functional requirements among the heterodimers formed by Mlh1p.
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http://dx.doi.org/10.1534/genetics.110.116806DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2881129PMC
June 2010

Interaction of genetic and environmental factors in Saccharomyces cerevisiae meiosis: the devil is in the details.

Methods Mol Biol 2009 ;557:3-20

Department of Genetics, University of Leicester, Leicester, United Kingdom.

One of the most important principles of scientific endeavour is that the results be reproducible from lab to lab. Although research groups rarely redo the published experiments of their colleagues, research plans almost always rely on the work of someone else. The assumption is that if the same experiment were repeated in another lab, results would be so similar that the same interpretation would be favoured. This notion allows one researcher to compare his/her own results to earlier work from other labs. An essential prerequisite for this is that the experiments are done in identical conditions and therefore the methodology must be clearly stated. While this may be scientific common sense, adherence is difficult because "standard" methods vary from one laboratory to another in subtle ways that are often not reported. More importantly, for many years the field ofyeast meiotic recombination considered typical differences to be innocuous. This chapter will highlight the documented environmental and genetic variables that are known to influence meiotic recombination in Saccharomyces cerevisiae. Other potential methodological sources of variation in meiotic experiments are also discussed. A careful assessment of the effects of these variables, has led to insights into our understanding of the control of recombination and meiosis.
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http://dx.doi.org/10.1007/978-1-59745-527-5_1DOI Listing
November 2009
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