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1.
Elife ; 132024 May 30.
Article in English | MEDLINE | ID: mdl-38813868

ABSTRACT

Germline epigenetic programming, including genomic imprinting, substantially influences offspring development. Polycomb Repressive Complex 2 (PRC2) plays an important role in Histone 3 Lysine 27 trimethylation (H3K27me3)-dependent imprinting, loss of which leads to growth and developmental changes in mouse offspring. In this study, we show that offspring from mouse oocytes lacking the PRC2 protein Embryonic Ectoderm Development (EED) were initially developmentally delayed, characterised by low blastocyst cell counts and substantial growth delay in mid-gestation embryos. This initial developmental delay was resolved as offspring underwent accelerated fetal development and growth in late gestation resulting in offspring that were similar stage and weight to controls at birth. The accelerated development and growth in offspring from Eed-null oocytes was associated with remodelling of the placenta, which involved an increase in fetal and maternal tissue size, conspicuous expansion of the glycogen-enriched cell population, and delayed parturition. Despite placental remodelling and accelerated offspring fetal growth and development, placental efficiency, and fetal blood glucose levels were low, and the fetal blood metabolome was unchanged. Moreover, while expression of the H3K27me3-imprinted gene and amino acid transporter Slc38a4 was increased, fetal blood levels of individual amino acids were similar to controls, indicating that placental amino acid transport was not enhanced. Genome-wide analyses identified extensive transcriptional dysregulation and DNA methylation changes in affected placentas, including a range of imprinted and non-imprinted genes. Together, while deletion of Eed in growing oocytes resulted in fetal growth and developmental delay and placental hyperplasia, our data indicate a remarkable capacity for offspring fetal growth to be normalised despite inefficient placental function and the loss of H3K27me3-dependent genomic imprinting.


Subject(s)
Genomic Imprinting , Animals , Female , Pregnancy , Mice , Polycomb Repressive Complex 2/metabolism , Polycomb Repressive Complex 2/genetics , Fetal Development/genetics , Placenta/metabolism , Oocytes/metabolism , Oocytes/growth & development , Amino Acid Transport System A
2.
BMC Biol ; 21(1): 281, 2023 12 05.
Article in English | MEDLINE | ID: mdl-38053127

ABSTRACT

BACKGROUND: Disrupted germline differentiation or compromised testis development can lead to subfertility or infertility and are strongly associated with testis cancer in humans. In mice, SRY and SOX9 induce expression of Fgf9, which promotes Sertoli cell differentiation and testis development. FGF9 is also thought to promote male germline differentiation but the mechanism is unknown. FGFs typically signal through mitogen-activated protein kinases (MAPKs) to phosphorylate ERK1/2 (pERK1/2). We explored whether FGF9 regulates male germline development through MAPK by inhibiting either FGF or MEK1/2 signalling in the foetal testis immediately after gonadal sex determination and testis cord formation, but prior to male germline commitment. RESULTS: pERK1/2 was detected in Sertoli cells and inhibition of MEK1/2 reduced Sertoli cell proliferation and organisation and resulted in some germ cells localised outside of the testis cords. While pERK1/2 was not detected in germ cells, inhibition of MEK1/2 after somatic sex determination profoundly disrupted germ cell mitotic arrest, dysregulated a broad range of male germline development genes and prevented the upregulation of key male germline markers, DPPA4 and DNMT3L. In contrast, while FGF inhibition reduced Sertoli cell proliferation, expression of male germline markers was unaffected and germ cells entered mitotic arrest normally. While male germline differentiation was not disrupted by FGF inhibition, a range of stem cell and cancer-associated genes were commonly altered after 24 h of FGF or MEK1/2 inhibition, including genes involved in the maintenance of germline stem cells, Nodal signalling, proliferation, and germline cancer. CONCLUSIONS: Together, these data demonstrate a novel role for MEK1/2 signalling during testis development that is essential for male germline differentiation, but indicate a more limited role for FGF signalling. Our data indicate that additional ligands are likely to act through MEK1/2 to promote male germline differentiation and highlight a need for further mechanistic understanding of male germline development.


Subject(s)
Neoplasms , Testis , Male , Mice , Humans , Animals , Testis/metabolism , Fibroblast Growth Factor 2 , Germ Cells , Cell Differentiation , Neoplasms/metabolism
3.
Clin Epigenetics ; 14(1): 183, 2022 12 21.
Article in English | MEDLINE | ID: mdl-36544159

ABSTRACT

BACKGROUND: Non-genetic disease inheritance and offspring phenotype are substantially influenced by germline epigenetic programming, including genomic imprinting. Loss of Polycomb Repressive Complex 2 (PRC2) function in oocytes causes non-genetically inherited effects on offspring, including embryonic growth restriction followed by post-natal offspring overgrowth. While PRC2-dependent non-canonical imprinting is likely to contribute, less is known about germline epigenetic programming of non-imprinted genes during oocyte growth. In addition, de novo germline mutations in genes encoding PRC2 lead to overgrowth syndromes in human patients, but the extent to which PRC2 activity is conserved in human oocytes is poorly understood. RESULTS: In this study, we identify a discrete period of early oocyte growth during which PRC2 is expressed in mouse growing oocytes. Deletion of Eed during this window led to the de-repression of 343 genes. A high proportion of these were developmental regulators, and the vast majority were not imprinted genes. Many of the de-repressed genes were also marked by the PRC2-dependent epigenetic modification histone 3 lysine 27 trimethylation (H3K27me3) in primary-secondary mouse oocytes, at a time concurrent with PRC2 expression. In addition, we found H3K27me3 was also enriched on many of these genes by the germinal vesicle (GV) stage in human oocytes, strongly indicating that this PRC2 function is conserved in the human germline. However, while the 343 genes were de-repressed in mouse oocytes lacking EED, they were not de-repressed in pre-implantation embryos and lost H3K27me3 during pre-implantation development. This implies that H3K27me3 is a transient feature that represses a wide range of genes in oocytes. CONCLUSIONS: Together, these data indicate that EED has spatially and temporally distinct functions in the female germline to repress a wide range of developmentally important genes and that this activity is conserved in the mouse and human germlines.


Subject(s)
DNA Methylation , Histones , Oocytes , Polycomb Repressive Complex 2 , Animals , Mice , Genes, Developmental , Histones/metabolism , Oocytes/growth & development , Oocytes/metabolism , Polycomb Repressive Complex 2/genetics , Polycomb Repressive Complex 2/metabolism
4.
Front Endocrinol (Lausanne) ; 13: 896747, 2022.
Article in English | MEDLINE | ID: mdl-35721752

ABSTRACT

Activin A, a TGFß superfamily member, is important for normal testis development through its actions on Sertoli cell development. Our analyses of altered activin A mouse models indicated gonocyte abnormalities, implicating activin A as a key determinant of early germline formation. Whether it acts directly or indirectly on germ cells is not understood. In humans, the fetal testis may be exposed to abnormally elevated activin A levels during preeclampsia, maternal infections, or following ingestion of certain medications. We hypothesized that this may impact fetal testis development and ultimately affect adult fertility. Germ cells from two mouse models of altered activin bioactivity were analysed. RNA-Seq of gonocytes purified from E13.5 and E15.5 Inhba KO mice (activin A subunit knockout) identified 46 and 44 differentially expressed genes (DEGs) respectively, and 45 in the E13.5 Inha KO (inhibin alpha subunit knockout; increased activin A) gonocytes. To discern direct effects of altered activin bioactivity on germline transcripts, isolated E13.5 gonocytes were cultured for 24h with activin A or with the activin/Nodal/TGFß inhibitor, SB431542. Gonocytes responded directly to altered signalling, with activin A promoting a more differentiated transcript profile (increased differentiation markers Dnmt3l, Nanos2 and Piwil4; decreased early germ cell markers Kit and Tdgf1), while SB431542 had a reciprocal effect (decreased Nanos2 and Piwil4; increased Kit). To delineate direct and indirect effects of activin A exposure on gonocytes, whole testes were cultured 48h with activin A or SB431542 and collected for histological and transcript analyses, or EdU added at the end of culture to measure germ and Sertoli cell proliferation using flow cytometry. Activin increased, and SB431542 decreased, Sertoli cell proliferation. SB431542-exposure resulted in germ cells escaping mitotic arrest. Analysis of FACS-isolated gonocytes following whole testis culture showed SB431542 increased the early germ cell marker Kit, however there was a general reduction in the impact of altered activin A bioavailability in the normal somatic cell environment. This multifaceted approach identifies a capacity for activin A to directly influence fetal germ cell development, highlighting the potential for altered activin A levels in utero to increase the risk of testicular pathologies that arise from impaired germline maturation.


Subject(s)
Activins , Germ Cells , Activins/metabolism , Animals , Argonaute Proteins , Germ Cells/metabolism , Male , Mice , RNA-Binding Proteins , Testis , Transforming Growth Factor beta
5.
Reproduction ; 163(3): 167-182, 2022 02 17.
Article in English | MEDLINE | ID: mdl-35084365

ABSTRACT

Polycomb repressive complex 2 (PRC2) catalyses the repressive epigenetic modification of histone 3 lysine 27 tri-methylation (H3K27me3) and functions as a key epigenetic regulator during embryonic development. PRC2 is known to regulate the development of a range of tissues by transcriptional silencing of genes that control cell differentiation, but its roles in female germline and ovarian development remain unknown. Using a mouse model with hypomorphic embryonic ectoderm development (EED) function that reduced H3K27me3 in somatic and germ cells, we found that PRC2 was required for survival, with more than 95% of female animals dying before birth. Although surviving adult EED hypomorphic females appeared morphologically similar to controls and were fertile, Eedhypo/hypo adult ovaries were abnormal, with altered morphology characterised by abnormal follicles. Early Eedhypo/hypo and control fetal ovaries were morphologically similar, and germ cells entered meiosis normally. Immunofluorescent analyses of somatic and germline markers indicated that ovarian development in Eedhypo/hypo ovaries was similar to heterozygous and WT controls. However, TUNEL analyses revealed higher rates of apoptosis in the ovarian surface epithelium, and transcriptional analyses revealed changes in genes regulating epithelial and steroidogenic cell differentiation, possibly foreshadowing the defects observed in adult ovaries of hypomorphic females. While it was possible to analyse early-mid fetal ovarian development, postnatal stages were inaccessible due to the high level of lethality during late fetal stages. Despite this limitation, the data we were able to obtain reveal a novel role for EED in the ovary that is likely to alter ovarian development and ovarian function in adult animals.


Subject(s)
Ovary , Polycomb Repressive Complex 2 , Animals , Cell Differentiation/genetics , Female , Histones/metabolism , Methylation , Mice , Ovary/metabolism , Polycomb Repressive Complex 2/genetics , Polycomb Repressive Complex 2/metabolism
6.
BMC Cancer ; 20(1): 960, 2020 Oct 06.
Article in English | MEDLINE | ID: mdl-33023532

ABSTRACT

BACKGROUND: The metzincin family of metalloproteinases and the tissue inhibitors of metalloproteinases (TIMPs) are essential proteins required for biological processes during cancer progression. This study aimed to determine the role of TIMP-2 in ovarian cancer progression and chemoresistance by reducing TIMP-2 expression in vitro in Fallopian tube secretory epithelial (FT282) and ovarian cancer (JHOS2 and OVCAR4) cell lines. METHODS: FT282, JHOS2 and OVCAR4 cells were transiently transfected with either single or pooled TIMP-2 siRNAs. The expression of different genes after TIMP-2 knock down (T2-KD) or in response to chemotherapy was determined at the mRNA level by quantitative real time PCR (qRT-PCR) and at the protein level by immunofluorescence. Sensitivity of the cell lines in response to chemotherapy after TIMP-2 knock down was investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 5-Ethynyl-2'-deoxyuridine (EdU) assays. Cell invasion in response to TIMP-2 knockdown was determined by xCELLigence. RESULTS: Sixty to 90 % knock down of TIMP-2 expression was confirmed in FT282, OVCAR4 and JHOS2 cell lines at the mRNA and protein levels. TIMP-2 knock down did not change the mRNA expression of TIMP-1 or TIMP-3. However, a significant downregulation of MMP-2 in T2-KD cells occurred at both the protein and activation levels, compared to Control (Cont; scrambled siRNA) and Parental cells (P, transfection reagent only). In contrast, membrane bound MT1-MMP protein levels were significantly upregulated in T2-KD compared to Cont and P cells. T2-KD cells exhibited enhanced proliferation and increased sensitivity to cisplatin and paclitaxel treatments. Enhanced invasion was observed in the T2-KD-JOSH2 and OVCAR4 cells but not in T2-KD-FT282 cells. Treatment with cisplatin or paclitaxel significantly elevated the expression of TIMP-2 in Cont cells but not in T2-KD cells, consistent with significantly elevated expression of chemoresistance and CSC markers and activation of STAT3. Furthermore, a potent inhibitor of STAT3 activation, Momelotinib, suppressed chemotherapy-induced activation of P-STAT3 in OVCAR4 cells with concomitant reductions in the expression of chemoresistance genes and CSC markers. CONCLUSIONS: The above results suggest that TIMP-2 may have a novel role in ovarian cancer proliferation, invasion and chemoresistance.


Subject(s)
Neoplastic Stem Cells/metabolism , Ovarian Neoplasms/metabolism , STAT3 Transcription Factor/metabolism , Tissue Inhibitor of Metalloproteinase-2/metabolism , Antineoplastic Agents, Phytogenic/pharmacology , Benzamides/pharmacology , Cell Line, Tumor , Cell Proliferation/physiology , Cystadenocarcinoma, Serous/drug therapy , Cystadenocarcinoma, Serous/genetics , Cystadenocarcinoma, Serous/metabolism , Cystadenocarcinoma, Serous/pathology , Drug Resistance, Neoplasm , Female , Humans , Neoplasm Invasiveness , Neoplastic Stem Cells/drug effects , Neoplastic Stem Cells/pathology , Ovarian Neoplasms/drug therapy , Ovarian Neoplasms/genetics , Ovarian Neoplasms/pathology , Paclitaxel/pharmacology , Protein Kinase Inhibitors/pharmacology , Pyrimidines/pharmacology , STAT3 Transcription Factor/antagonists & inhibitors , Transfection
7.
Pharmacol Res Perspect ; 8(5): e00654, 2020 10.
Article in English | MEDLINE | ID: mdl-32930516

ABSTRACT

There remains a compelling need for the development of nonsurgical sterilizing agents to expand the fertility management options for both domestic and feral animal species. We hypothesize that an efficacious sterilization approach would be to selectively ablate nonrenewable cell types that are essential for reproduction, such as the undifferentiated gonocytes within the embryonic gonad. Here, we report a novel strategy to achieve this goal centered on the use of a chemically modified M13 bacteriophage to effect the targeted delivery of menadione, a redox-cycling naphthoquinone, to mouse gonocytes. Panning of the M13 random peptide 'phage display library proved effective in the isolation of gonocyte-specific targeting clones. One such clone was modified via N-succinimidyl-S-acetylthioacetate (SATA) linkage to the N-terminus of the major PVIII capsid protein. Subsequent deacetylation of the SATA was undertaken to expose a thiol group capable of reacting with menadione through Michael addition. This chemical modification was confirmed using UV spectrophotometry. In proof-of-concept experiments we applied the modified 'phage to primary cultures of fetal germ cells and induced, an approximately, 60% reduction in the viability of the target cell population. These studies pave the way for in vivo application of chemically modified M13 bacteriophage in order to achieve the selective ablation of nonrenewable cell types in the reproductive system, thereby providing a novel nonsurgical approach the regulation of fertility in target species.


Subject(s)
Bacteriophage M13/physiology , Germ Cells/cytology , Sterilization, Reproductive/veterinary , Succinimides/chemistry , Sulfides/chemistry , Vitamin K 3/pharmacology , Animals , Bacteriophage M13/chemistry , Capsid Proteins/chemistry , Capsid Proteins/metabolism , Cell Survival/drug effects , Female , Germ Cells/drug effects , Male , Mice , Ovary/cytology , Ovary/drug effects , Peptide Library , Proof of Concept Study , Testis/cytology , Testis/drug effects
8.
Reproduction ; 160(1): 141-154, 2020 07.
Article in English | MEDLINE | ID: mdl-32484162

ABSTRACT

Disrupted fetal germline development underpins testicular germ cell neoplasia, which is increasing worldwide. The complex signaling milieu during normal testis development includes TGFß superfamily ligands; this study tests the hypothesis that, activin A, a TGFß superfamily member, can influence gonocyte development. The human seminoma-derived cell line, TCam-2, a model of fetal gonocytes, was cultured with activin A (1.25-25 ng/mL) for 48 h, or with 5 ng/mL activin A for short- (6, 24, and 48 h) and long-term (13 days) exposures, and downstream targets measured by qRT-PCR. Transcripts that exhibited significant dose-dependent responses to activin A included the early germ cell markers KIT, NODAL, and CRIPTO (NODALl co-receptor and activin inhibitor) which all increased and the differentiation marker DNMT3L which decreased. After 48 h, KIT, NODAL, and CRIPTO levels were significantly higher, while the differentiation marker NANOS2 was significantly lower. Interestingly, activin A exposure also significantly reduced both transcript and protein levels of the PIWI/piRNA pathway component DNMT3L. Because TCam-2 cells produce the activin inhibitor CRIPTO, CRIPTO was reduced using siRNA prior to activin A exposure. This selectively increased KIT in response to activin A. Other ligands present in the fetal testis (BMP4, FGF9, TGFß1, and TGFß2) induced distinct effects on germline marker expression. This study showed that activin A can directly modulate germline markers in this human gonocyte-like cell, promoting a less-differentiated phenotype. Additional findings indicate evidence of signaling crosstalk between activin A and NODAL, leading to target-specific effects on gonocyte differentiation.


Subject(s)
Activins/pharmacology , Cell Differentiation , Gene Expression Regulation/drug effects , Germ Cells/pathology , Nodal Protein/metabolism , Seminoma/pathology , Transforming Growth Factor beta/pharmacology , Gene Expression Profiling , Germ Cells/metabolism , Humans , Male , Nodal Protein/genetics , Seminoma/drug therapy , Seminoma/genetics , Testicular Neoplasms/drug therapy , Testicular Neoplasms/genetics , Testicular Neoplasms/pathology
9.
J Proteomics ; 223: 103830, 2020 07 15.
Article in English | MEDLINE | ID: mdl-32450490

ABSTRACT

Posttranslational modification by ubiquitination targets proteins for degradation, recycling, stabilization or altered trafficking, and as such can alter cellular signaling pathways. The substrate specificity of this multistep process is controlled by ubiquitin ligases, including those of the HECT domain-containing NEDD4 family. In the testis, ubiquitination of many proteins contributes to organ development and maturation of spermatozoa and NEDD4 is known to be important in the control of spermatogonial stem cell homeostasis. However, a comprehensive understanding of NEDD4 substrates in testis development is lacking. Here we demonstrate high expression of Nedd4 in somatic cells of the mouse testis and in the murine Leydig cell-like cell line TM3. Immunoprecipitation of NEDD4 tagged with GFP at either the amino or carboxyl terminus was subjected to proteomic analysis for interacting proteins. We identified a substantial list of potential interaction partners, including known NEDD4 substrates, proteins involved in ubiquitination and proteins important for testis development and spermatogenesis. We confirmed the interaction of NEDD4 with a subset of these putative interacting proteins, validating the integrity of the dataset. These potential interactors may be further explored to reveal important roles of NEDD4-mediated ubiquitination in the testis. SIGNIFICANCE: Ubiquitination is important for testis development and function, and NEDD4 is known to ubiquitinate various proteins to affect cellular signaling and development, including those implicated in spermatogenesis. However, substrates of NEDD4 that are important during testis development remain to be identified. Here we report NEDD4 expression in the developing testis and TM3 testicular cell line. This study identifies a substantial list of NEDD4 interacting proteins in the TM3 testicular cell line, with validation of some of these interactions. Hence, this provides novel NEDD4 targets that may contribute to testis development and function that may be further explored.


Subject(s)
Endosomal Sorting Complexes Required for Transport , Ubiquitin , Animals , Endosomal Sorting Complexes Required for Transport/genetics , Endosomal Sorting Complexes Required for Transport/metabolism , Male , Mice , Nedd4 Ubiquitin Protein Ligases , Proteomics , Testis/metabolism , Ubiquitin-Protein Ligases/metabolism , Ubiquitination
10.
Endocrinology ; 161(7)2020 07 01.
Article in English | MEDLINE | ID: mdl-32274496

ABSTRACT

Activin A promotes fetal mouse testis development, including driving Sertoli cell proliferation and cord morphogenesis, but its mechanisms of action are undefined. We performed ribonucleic acid sequencing (RNA-seq) on testicular somatic cells from fetal activin A-deficient mice (Inhba KO) and wildtype littermates at embryonic day (E) E13.5 and E15.5. Analysis of whole gonads provided validation, and cultures with a pathway inhibitor discerned acute from chronic effects of altered activin A bioactivity. Activin A deficiency predominantly affects the Sertoli cell transcriptome. New candidate targets include Minar1, Sel1l3, Vnn1, Sfrp4, Masp1, Nell1, Tthy1 and Prss12. Importantly, the testosterone (T) biosynthetic enzymes present in fetal Sertoli cells, Hsd17b1 and Hsd17b3, were identified as activin-responsive. Activin-deficient testes contained elevated androstenedione (A4), displayed an Inhba gene dose-dependent A4/T ratio, and contained 11-keto androgens. The remarkable accumulation of lipid droplets in both Sertoli and germ cells at E15.5 indicated impaired lipid metabolism in the absence of activin A. This demonstrated for the first time that activin A acts on Sertoli cells to determine local steroid production during fetal testis development. These outcomes reveal how compounds that perturb fetal steroidogenesis can function through cell-specific mechanisms and can indicate how altered activin levels in utero may impact testis development.


Subject(s)
Activins/physiology , Gonadal Steroid Hormones/metabolism , Testis/embryology , Testis/metabolism , Animals , Female , Male , Mice , Mice, Knockout , Pregnancy , Sex Determination Processes
12.
BMC Biol ; 16(1): 104, 2018 09 20.
Article in English | MEDLINE | ID: mdl-30236109

ABSTRACT

BACKGROUND: Defining the mechanisms that establish and regulate the transmission of epigenetic information from parent to offspring is critical for understanding disease heredity. Currently, the molecular pathways that regulate epigenetic information in the germline and its transmission to offspring are poorly understood. RESULTS: Here we provide evidence that Polycomb Repressive Complex 2 (PRC2) regulates paternal inheritance. Reduced PRC2 function in mice resulted in male sub-fertility and altered epigenetic and transcriptional control of retrotransposed elements in foetal male germ cells. Males with reduced PRC2 function produced offspring that over-expressed retrotransposed pseudogenes and had altered preimplantation embryo cleavage rates and cell cycle control. CONCLUSION: This study reveals a novel role for the histone-modifying complex, PRC2, in paternal intergenerational transmission of epigenetic effects on offspring, with important implications for understanding disease inheritance.


Subject(s)
Epigenesis, Genetic/genetics , Germ Cells/metabolism , Paternal Inheritance/genetics , Polycomb Repressive Complex 2/genetics , Animals , Male , Mice , Polycomb Repressive Complex 2/metabolism
13.
Clin Epigenetics ; 10(1): 95, 2018 07 13.
Article in English | MEDLINE | ID: mdl-30005706

ABSTRACT

BACKGROUND: Investigating how epigenetic information is transmitted through the mammalian germline is the key to understanding how this information impacts on health and disease susceptibility in offspring. EED is essential for regulating the repressive histone modification, histone 3 lysine 27 tri-methylation (H3K27me3) at many developmental genes. RESULTS: In this study, we used oocyte-specific Zp3-Cre recombinase (Zp3Cre) to delete Eed specifically in mouse growing oocytes, permitting the study of EED function in oocytes and the impact of depleting EED in oocytes on outcomes in offspring. As EED deletion occurred only in growing oocytes and females were mated to normal wild type males, this model allowed the study of oocyte programming without confounding factors such as altered in utero environment. Loss of EED from growing oocytes resulted in a significant overgrowth phenotype that persisted into adult life. Significantly, this involved increased adiposity (total fat) and bone mineral density in offspring. Similar overgrowth occurs in humans with Cohen-Gibson (OMIM 617561) and Weaver (OMIM 277590) syndromes, that result from de novo germline mutations in EED or its co-factor EZH2, respectively. Consistent with a role for EZH2 in human oocytes, we demonstrate that de novo germline mutations in EZH2 occurred in the maternal germline in some cases of Weaver syndrome. However, deletion of Ezh2 in mouse oocytes resulted in a distinct phenotype compared to that resulting from oocyte-specific deletion of Eed. CONCLUSIONS: This study provides novel evidence that altering EED-dependent oocyte programming leads to compromised offspring growth and development in the next generation.


Subject(s)
Gene Deletion , Growth Disorders/genetics , Oocytes/growth & development , Polycomb Repressive Complex 2/genetics , Adiposity , Animals , Bone Density , Disease Models, Animal , Enhancer of Zeste Homolog 2 Protein/genetics , Female , Growth Disorders/metabolism , Humans , Male , Maternal Inheritance , Mice , Oocytes/metabolism
14.
Clin Epigenetics ; 10: 33, 2018.
Article in English | MEDLINE | ID: mdl-29515677

ABSTRACT

Background: Recently discovered drugs that target epigenetic modifying complexes are providing new treatment options for a range of cancers that affect patients of reproductive age. Although these drugs provide new therapies, it is likely that they will also affect epigenetic programming in sperm and oocytes. A promising target is Enhancer of Zeste 2 (EZH2), which establishes the essential epigenetic modification, H3K27me3, during development. Results: In this study, we demonstrate that inhibition of EZH1/2 with the clinically relevant drug, tazemetostat, severely depletes H3K27me3 in growing oocytes of adult female mice. Moreover, EZH2 inhibition depleted H3K27me3 in primary oocytes and in fetal oocytes undergoing epigenetic reprogramming. Surprisingly, once depleted, H3K27me3 failed to recover in growing oocytes or in fetal oocytes. Conclusion: Together, these data demonstrate that drugs targeting EZH2 significantly affect the germline epigenome and, based on genetic models with oocyte-specific loss of EZH2 function, are likely to affect outcomes in offspring.


Subject(s)
Benzamides/administration & dosage , Histones/metabolism , Oocytes/growth & development , Pyridones/administration & dosage , Animals , Benzamides/pharmacology , Biphenyl Compounds , Down-Regulation , Enhancer of Zeste Homolog 2 Protein/antagonists & inhibitors , Epigenesis, Genetic/drug effects , Female , Mice , Morpholines , Oocytes/cytology , Oocytes/drug effects , Oocytes/metabolism , Organ Culture Techniques , Pyridones/pharmacology
15.
Mol Cell Endocrinol ; 468: 121-133, 2018 06 15.
Article in English | MEDLINE | ID: mdl-29471014

ABSTRACT

The testis and ovary provide specialised environments that nurture germ cells and facilitate their maturation, culminating in the production of mature gametes that can found the following generation. The sperm and egg not only transmit genetic information, but also epigenetic modifications that affect the development and physiology of offspring. Importantly, the epigenetic information contained in mature sperm and oocytes can be influenced by a range of environmental factors, such as diet, chemicals and drugs. An increasing range of studies are revealing how gene-environment interactions are mediated through the germline. Outside the germline, altered epigenetic state is common in a range of diseases, including many cancers. As epigenetic modifications are reversible, pharmaceuticals that directly target epigenetic modifying proteins have been developed and are delivering substantial benefits to patients, particularly in oncology. While providing the most effective patient treatment is clearly the primary concern, some patients will want to conceive children after treatment. However, the impacts of epigenomic drugs on the male and female gametes are poorly understood and whether these drugs will have lasting effects on patients' germline epigenome and subsequent offspring remains largely undetermined. Currently, evidence based clinical guidelines for use of epigenomic drugs in patients of reproductive age are limited in this context. Developing a deeper understanding of the epigenetic mechanisms regulating the germline epigenome and its impact on inherited traits and disease susceptibility is required to determine how specific epigenomic drugs might affect the germline and inheritance. Understanding these potential effects will facilitate the development of informed clinical guidelines appropriate for the use of epigenomic drugs in patients of reproductive age, ultimately improving the safety of these therapies in the clinic.


Subject(s)
Epigenomics , Germ Cells/metabolism , Inheritance Patterns/genetics , Animals , Blastocyst/metabolism , Cellular Reprogramming/genetics , Guidelines as Topic , Humans
16.
PLoS One ; 13(1): e0190925, 2018.
Article in English | MEDLINE | ID: mdl-29324788

ABSTRACT

ESRP1 regulates alternative splicing, producing multiple transcripts from its target genes in epithelial tissues. It is upregulated during mesenchymal to epithelial transition associated with reprogramming of fibroblasts to iPS cells and has been linked to pluripotency. Mouse fetal germ cells are the founders of the adult gonadal lineages and we found that Esrp1 mRNA was expressed in both male and female germ cells but not in gonadal somatic cells at various stages of gonadal development (E12.5-E15.5). In the postnatal testis, Esrp1 mRNA was highly expressed in isolated cell preparations enriched for spermatogonia but expressed at lower levels in those enriched for pachytene spermatocytes and round spermatids. Co-labelling experiments with PLZF and c-KIT showed that ESRP1 was localized to nuclei of both Type A and B spermatogonia in a speckled pattern, but was not detected in SOX9+ somatic Sertoli cells. No co-localization with the nuclear speckle marker, SC35, which has been associated with post-transcriptional splicing, was observed, suggesting that ESRP1 may be associated with co-transcriptional splicing or have other functions. RNA interference mediated knockdown of Esrp1 expression in the seminoma-derived Tcam-2 cell line demonstrated that ESRP1 regulates alternative splicing of mRNAs in a non-epithelial cell germ cell tumour cell line.


Subject(s)
Germ Cells/metabolism , RNA-Binding Proteins/metabolism , Spermatogenesis/physiology , Testis/growth & development , Testis/metabolism , Alternative Splicing , Animals , Cell Line, Tumor , Cells, Cultured , Female , Gene Expression , Germ Cells/cytology , Male , Mice, Inbred C57BL , RNA, Messenger/metabolism , Testis/cytology
17.
F1000Res ; 72018.
Article in English | MEDLINE | ID: mdl-30613387

ABSTRACT

Epigenetic modifications, including DNA methylation and histone modifications, determine the way DNA is packaged within the nucleus and regulate cell-specific gene expression. The heritability of these modifications provides a memory of cell identity and function. Common dysregulation of epigenetic modifications in cancer has driven substantial interest in the development of epigenetic modifying drugs. Although these drugs have the potential to be highly beneficial for patients, they act systemically and may have "off-target" effects in other cells such as the patients' sperm or eggs. This review discusses the potential for epigenomic drugs to impact on the germline epigenome and subsequent offspring and aims to foster further examination into the possible effects of these drugs on gametes. Ultimately, the information gained by further research may improve the clinical guidelines for the use of such drugs in patients of reproductive age.


Subject(s)
Drug-Related Side Effects and Adverse Reactions/genetics , Epigenomics/methods , Germ Cells/drug effects , DNA Methylation , Histone Code , Humans , Reproduction/drug effects
18.
Article in English | MEDLINE | ID: mdl-28239420

ABSTRACT

BACKGROUND: Defining how epigenetic information is established in the germline during fetal development is key to understanding how epigenetic information is inherited and impacts on evolution and human health and disease. RESULTS: Here, we show that Polycomb Repressive Complex 2 is transiently localized in the nucleus of mouse fetal germ cells, while DNA methylation is removed from the germline. This coincides with significant enrichment of trimethylated lysine 27 on histone 3 near the nuclear lamina that is dependent on activity of the essential PRC2 catalytic proteins, Enhancer of Zeste 1 and/or 2. CONCLUSIONS: Combined, these data reveal a role for Polycomb Repressive Complex 2 and trimethylated lysine 27 on histone 3 during germline epigenetic programming that we speculate is required to repress target sequences while DNA methylation is removed.


Subject(s)
Epigenomics , Histones/metabolism , Polycomb Repressive Complex 2/metabolism , Animals , Cell Differentiation , Cellular Reprogramming , Enhancer of Zeste Homolog 2 Protein/metabolism , Female , Fetus/cytology , Germ Cells/cytology , Germ Cells/drug effects , Germ Cells/metabolism , Gonads/metabolism , Gonads/pathology , Histones/genetics , Indoles/pharmacology , Male , Methylation , Mice , Mice, Transgenic , Microscopy, Fluorescence , Octamer Transcription Factor-3/genetics , Pyridones/pharmacology
19.
Reproduction ; 152(5): 529-43, 2016 11.
Article in English | MEDLINE | ID: mdl-27495231

ABSTRACT

Testis development is dependent on the key sex-determining factors SRY and SOX9, which activate the essential ligand FGF9. Although FGF9 plays a central role in testis development, it is unable to induce testis formation on its own. However, other growth factors, including activins and TGFßs, also present testis during testis formation. In this study, we investigated the potential of FGF9 combined with activin and TGFß to induce testis development in cultured XX gonads. Our data demonstrated differing individual and combined abilities of FGF9, activin and TGFß to promote supporting cell proliferation, Sertoli cell development and male germ line differentiation in cultured XX gonads. FGF9 promoted proliferation of supporting cells in XX foetal gonads at rates similar to those observed in vivo during testis cord formation in XY gonads but was insufficient to initiate testis development. However, when FGF9, activin and TGFß were combined, aspects of testicular development were induced, including the expression of Sox9, morphological reorganisation of the gonad and deposition of laminin around germ cells. Enhancing ß-catenin activity diminished the testis-promoting activities of the combined growth factors. The male promoting activity of FGF9 and the combined growth factors directly or indirectly extended to the germ line, in which a mixed phenotype was observed. FGF9 and the combined growth factors promoted male germ line development, including mitotic arrest, but expression of pluripotency genes was maintained, rather than being repressed. Together, our data provide evidence that combined signalling by FGF9, activin and TGFß can induce testicular characteristics in XX gonads.


Subject(s)
Fibroblast Growth Factor 9/metabolism , Inhibin-beta Subunits/metabolism , Organ Culture Techniques/methods , Ovary/cytology , Testis/cytology , Transforming Growth Factor beta/metabolism , Animals , Female , Fibroblast Growth Factor 9/genetics , Inhibin-beta Subunits/genetics , Male , Mice , Ovary/metabolism , Sex Differentiation , Signal Transduction , Testis/metabolism , Transforming Growth Factor beta/genetics , beta Catenin/genetics , beta Catenin/metabolism
20.
Semin Immunopathol ; 38(6): 765-781, 2016 11.
Article in English | MEDLINE | ID: mdl-27315198

ABSTRACT

Male gamete development begins with the specification of primordial cells in the epiblast of the early embryo and is not complete until spermatozoa mature in the epididymis of adult males. This protracted developmental process involves extensive alteration of the paternal germline epigenome. Initially, epigenetic reprogramming in fetal germ cells results in removal of most DNA methylation, including parent-specific epigenetic information. The germ cells then establish sex-specific epigenetic information through de novo methylation and undergo spermatogenesis. Chromatin in haploid germ cells is repackaged into protamines during spermiogenesis, providing further widespread epigenetic reorganization. Finally, after fertilization, epigenetic reprogramming in the preimplantation embryo is necessary for regaining totipotency. These events provide substantial windows during which epigenetic errors either may be corrected or may occur in the germline. There is now increasing evidence that environmental factors such as exposure to toxicants, the parents' and individual's diet, and even infectious and inflammatory events in the male reproductive tract may influence epigenetic reprogramming. This, together with other damage inflicted on the germline chromatin, may result in negative consequences for fertility and health. Large epidemiological birth cohort studies have yielded insight into possible causative environmental factors. Together with experimental animal studies, a clearer view of environmental impacts on fetal development and their intergenerational and even transgenerational effects on reproductive health has emerged and is reviewed in this article.


Subject(s)
Infertility, Male/etiology , Prenatal Exposure Delayed Effects , Biomarkers , Cell Differentiation , Cellular Reprogramming , Environment , Epididymis/embryology , Epididymis/immunology , Epigenesis, Genetic , Female , Gene Expression Regulation, Developmental , Gene-Environment Interaction , Germ Cells/cytology , Germ Cells/metabolism , Humans , Infections/complications , Inflammation/complications , Male , Maternal Exposure/adverse effects , Pregnancy , Risk Factors , Testis/embryology , Testis/immunology
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