RESUMO
Oocytes in embryonic ovaries enter meiosis I and arrest in the diplonema stage. Perturbations in meiosis I, such as abnormal double-strand break (DSB) formation and repair, adversely affect oocyte survival. We previously discovered that HORMAD1 is a critical component of the synaptonemal complex but not essential for oocyte survival. No significant differences were observed in the number of primordial, primary, secondary, and developing follicles between wild-type and Hormad1(−/−)newborn, 8-day, and 80-day ovaries. Meiosis I progression in Hormad1(−/−) embryonic ovaries was normal through the zygotene stage and in oocytes arrested in diplonema; however, we did not visualize oocytes with completely synapsed chromosomes. We investigated effects of HORMAD1 deficiency on the kinetics of DNA DSB formation and repair in the mouse ovary. We irradiated Embryonic Day 16.5 wild-type and Hormad1(−/−) ovaries and monitored DSB repair using gammaH2AX, RAD51, and DMC1 immunofluorescence. Our results showed a significant drop in unrepaired DSBs in the irradiated Hormad1(−/−) zygotene oocytes as compared to the wild-type oocytes. Moreover, Hormad1 deficiency rescued Dmc1(−/−) oocytes. These results indicate that Hormad1 deficiency promotes DMC1-independent DSB repairs, which in turn helps asynaptic Hormad1(−/−) oocytes resist perinatal loss.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA/genética , Meiose/genética , Animais , Pontos de Checagem do Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , Feminino , Camundongos , Camundongos Knockout , Oócitos/metabolismoRESUMO
OBJECTIVE: To examine gonadal protective properties of granulocyte colony-stimulating factor (G-CSF) alone or in combination with stem cell factor (SCF) in female mice treated with high-dose alkylating chemotherapy. DESIGN: Experimental laboratory animal study. SETTING: Tertiary care academic hospital and research institute. ANIMAL(S): Six- and 8-week-old C57Bl/6 female mice. INTERVENTION(S): Adult female mice were treated with [1] cyclophosphamide and busulfan (CTx), [2] CTx + G-CSF/SCF, [3] CTx + G-CSF, or [4] normal saline and dimethyl sulfoxide (DMSO; vehicle control). MAIN OUTCOME MEASURE(S): Follicle counts, microvessel density, cellular response to DNA damage, and litter production. RESULT(S): G-CSF ± SCF increased microvessel density and decreased follicle loss in CTx-treated female mice compared with CTx-only treated female mice. Mice administered CTx alone exhibited premature ovarian insufficiency, with only 28% of mice producing two litters. However, 100% of mice receiving CTx with G-CSF + SCF, and 80% of mice receiving CTx + G-CSF alone produced at least three litters and 20% of mice in each group produced five litters. CONCLUSION(S): Treatment of mice with G-CSF decreases chemotherapy-induced ovarian follicle loss and extends time to premature ovarian insufficiency in female mice. Further studies are needed to validate these preclinical results in humans and compare efficacy with the established GnRH analogue treatments.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica , Fármacos para a Fertilidade Feminina/farmacologia , Preservação da Fertilidade/métodos , Fertilidade/efeitos dos fármacos , Fator Estimulador de Colônias de Granulócitos/farmacologia , Infertilidade Feminina/prevenção & controle , Ovário/efeitos dos fármacos , Insuficiência Ovariana Primária/prevenção & controle , Fator de Células-Tronco/farmacologia , Animais , Bussulfano , Ciclofosfamida , Dano ao DNA , Modelos Animais de Doenças , Quimioterapia Combinada , Feminino , Filgrastim , Histonas/metabolismo , Infertilidade Feminina/induzido quimicamente , Infertilidade Feminina/fisiopatologia , Tamanho da Ninhada de Vivíparos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Microvasos/efeitos dos fármacos , Folículo Ovariano/efeitos dos fármacos , Folículo Ovariano/fisiopatologia , Ovário/irrigação sanguínea , Ovário/fisiopatologia , Paridade/efeitos dos fármacos , Insuficiência Ovariana Primária/induzido quimicamente , Insuficiência Ovariana Primária/fisiopatologia , Proteínas Recombinantes/farmacologia , Fatores de TempoRESUMO
Uterine leiomyomas (uterine fibroids) arise from smooth muscle tissue in the majority of women by age 45. It is common for these clonal tumors to develop from multiple locations within the uterus, leading to a variety of symptoms such as pelvic pain, abnormal uterine bleeding, and infertility. We performed whole exome sequencing on genomic DNA from five pairs of leiomyomas and corresponding normal myometrium to determine genetic variations unique to leiomyomas. Whole exome sequencing revealed that the gene encoding transcription factor MED12 (Mediator complex subunit 12) harbored heterozygous missense mutations caused by single nucleotide variants in highly conserved codon 44 of exon 2 in two of five leiomyomas. Sanger re-sequencing of MED12 among these five leiomyomas confirmed the two single nucleotide variants and detected a 42 base-pair deletion within exon 2 of MED12 in a third leiomyoma. MED12 was sequenced in an additional 143 leiomyomas and 73 normal myometrial tissues. Overall, MED12 was mutated in 100/148 (67%) of the genotyped leiomyomas: 79/148 (53%) leiomyomas exhibited heterozygous missense single nucleotide variants, 17/148 (11%) leiomyomas exhibited heterozygous in-frame deletions/insertion-deletions, 2/148 (1%) leiomyomas exhibited intronic heterozygous single nucleotide variants affecting splicing, and 2/148 (1%) leiomyomas exhibited heterozygous deletions/insertion-deletions spanning the intron 1-exon 2 boundary which affected the splice acceptor site. Mutations were not detected in MED12 in normal myometrial tissue. MED12 mutations were equally distributed among karyotypically normal and abnormal uterine leiomyomas and were identified in leiomyomas from both black and white American women. Our studies show an association between MED12 mutations and leiomyomas in ethnically and racially diverse American women.
Assuntos
Predisposição Genética para Doença/genética , Leiomiomatose/epidemiologia , Leiomiomatose/genética , Complexo Mediador/genética , Mutação/genética , Neoplasias Uterinas/epidemiologia , Neoplasias Uterinas/genética , Sequência de Bases , DNA Complementar/genética , Exoma/genética , Feminino , Estudos de Associação Genética , Humanos , Dados de Sequência Molecular , América do Norte/epidemiologia , Análise de Sequência de DNARESUMO
Human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) have been shown to differentiate into primordial germ cells (PGCs) but not into spermatogonia, haploid spermatocytes, or spermatids. Here, we show that hESCs and hiPSCs differentiate directly into advanced male germ cell lineages, including postmeiotic, spermatid-like cells, in vitro without genetic manipulation. Furthermore, our procedure mirrors spermatogenesis in vivo by differentiating PSCs into UTF1-, PLZF-, and CDH1-positive spermatogonia-like cells; HIWI- and HILI-positive spermatocyte-like cells; and haploid cells expressing acrosin, transition protein 1, and protamine 1 (proteins that are uniquely found in spermatids and/or sperm). These spermatids show uniparental genomic imprints similar to those of human sperm on two loci: H19 and IGF2. These results demonstrate that male PSCs have the ability to differentiate directly into advanced germ cell lineages and may represent a novel strategy for studying spermatogenesis in vitro.
Assuntos
Diferenciação Celular/fisiologia , Haploidia , Células-Tronco Pluripotentes/metabolismo , Espermátides/metabolismo , Espermatócitos/metabolismo , Espermatogênese/fisiologia , Animais , Linhagem Celular , Humanos , Masculino , Camundongos , Células-Tronco Pluripotentes/citologia , Espermátides/citologia , Espermatócitos/citologia , Fatores de Transcrição/metabolismoRESUMO
OBJECTIVE: To analyze DNA from women with premature ovarian failure (POF) for genome-wide copy-number variations (CNVs), focusing on novel autosomal microdeletions. DESIGN: Case-control genetic association study. SETTING: Department of Obstetrics and Gynecology, Baylor College of Medicine, Houston, Texas. PATIENT(S): Of 89 POF patients, eight experienced primary amenorrhea and 81 exhibited secondary amenorrhea before age 40 years. INTERVENTION(S): Genomic DNA from peripheral blood samples was analyzed for CNVs using high-resolution single-nucleotide polymorphism (SNP) arrays. MAIN OUTCOME MEASURE(S): Identification of novel CNVs in 89 POF cases, using the Database of Genomic Variants as a control population. RESULT(S): A total of 198 autosomal CNVs were detected by SNP arrays, ranging in size from 0.1 Mb to 3.4 Mb. These CNVs (>0.1 Mb) included 17 novel microduplications and seven novel microdeletions, six of which contained the coding regions 8q24.13, 10p15-p14, 10q23.31, 10q26.3, 15q25.2, and 18q21.32. Most of the novel CNVs were derived from autosomes rather than the X chromosome. CONCLUSION(S): The present pilot study revealed novel microdeletions/microduplications in women with POF. Two novel microdeletions caused haploinsufficiency for SYCE1 and CPEB1, genes known to cause ovarian failure in knockout mouse models. Chromosomal microarrays may be a useful adjunct to conventional karyotyping when evaluating genomic imbalances in women with POF.