Jessica M. Stringer

Signaling through the TGF Beta-Activin Receptors ALK4/5/7 Regulates Testis Formation and Male Germ Cell Development

The developing testis provides an environment that nurtures germ cell development, ultimately ensuring spermatogenesis and fertility. Impacts on this environment are considered to underlie aberrant germ cell development and formation of germ cell tumour precursors. The signaling events involved in testis formation and male fetal germ cell development remain largely unknown. Analysis of knockout mice lacking single Tgfβ family members has indicated that Tgfβs are not required for sex determination. However, due to functional redundancy, it is possible that additional functions for these ligands in gonad development remain to be discovered. Using FACS purified gonadal cells, in this study we show that the genes encoding Activins, TGFβs, Nodal and their respective receptors, are expressed in sex and cell type specific patterns suggesting particular roles in testis and germ cell development. Inhibition of signaling through the receptors ALK4, ALK5 and ALK7, and ALK5 alone, demonstrated that TGFβ signaling is required for testis cord formation during the critical testis-determining period. We also show that signaling through the Activin/NODAL receptors, ALK4 and ALK7 is required for promoting differentiation of male germ cells and their entry into mitotic arrest. Finally, our data demonstrate that Nodal is specifically expressed in male germ cells and expression of the key pluripotency gene, Nanog was significantly reduced when signaling through ALK4/5/7 was blocked. Our strategy of inhibiting multiple Activin/NODAL/TGFβ receptors reduces the functional redundancy between these signaling pathways, thereby revealing new and essential roles for TGFβ and Activin signaling during testis formation and male germ cell development.

Fine-tuning evolution: germ-line epigenetics and inheritance

In mice, epiblast cells found both the germ-line and somatic lineages in the developing embryo. These epiblast cells carry epigenetic information from both parents that is required for development and cell function in the fetus and during post-natal life. However, germ cells must establish an epigenetic program that supports totipotency and the configuration of parent-specific epigenetic states in the gametes. To achieve this, the epigenetic information inherited by the primordial germ cells at specification is erased and new epigenetic states are established during development of the male and female germ-lines. Errors in this process can lead to transmission of epimutations through the germ-line, which have the potential to affect development and disease in the parents progeny. This review discusses epigenetic reprogramming in the germ-line and the transmission of epigenetic information to the following generation.

Transgenerational epigenetic inheritance: adaptation through the germline epigenome?

Epigenetic modifications direct the way DNA is packaged into the nucleus, making genes more or less accessible to transcriptional machinery and influencing genomic stability. Environmental factors have the potential to alter the epigenome, allowing genes that are silenced to be activated and vice versa. This ultimately influences disease susceptibility and health in an individual. Furthermore, altered chromatin states can be transmitted to subsequent generations, thus epigenetic modifications may provide evolutionary mechanisms that impact on adaptation to changed environments. However, the mechanisms involved in establishing and maintaining these epigenetic modifications during development remain unclear. This review discusses current evidence for transgenerational epigenetic inheritance, confounding issues associated with its study, and the biological relevance of altered epigenetic states for subsequent generations.

Selected imprinting of INS in the marsupial

BackgroundIn marsupials, growth and development of the young occur postnatally, regulated by milk that changes in composition throughout the long lactation. To initiate lactation in mammals, there is an absolute requirement for insulin (INS), a gene known to be imprinted in the placenta. We therefore examined whether INS is imprinted in the mammary gland of the marsupial tammar wallaby (Macropus eugenii) and compared its expression with that of insulin-like growth factor 2 (IGF2).ResultsINS was expressed in the mammary gland and significantly increased, while IGF2 decreased, during established milk production. Insulin and IGF2 were both detected in the mammary gland macrophage cells during early lactation and in the alveolar cells later in lactation. Surprisingly, INS, which was thought only to be imprinted in the therian yolk sac, was imprinted and paternally expressed in the liver of the developing young, monoallelically expressed in the tammar mammary gland and biallelic in the stomach and intestine. The INS transcription start site used in the liver and mammary gland was differentially methylated.ConclusionsThis is the first study to identify tissue-specific INS imprinting outside the yolk sac. These data suggest that there may be an advantage of selective monoallelic expression in the mammary gland and that this may influence the growth of the postnatal young. These results are not consistent with the parental conflict hypothesis, but instead provide support for the maternal–infant co-adaptation hypothesis. Thus, imprinting in the mammary gland maybe as critical for postnatal growth and development in mammals as genomic imprinting in the placenta is prenatally.

Post-natal imprinting: evidence from marsupials.

Genomic imprinting has been identified in therian (eutherian and marsupial) mammals but not in prototherian (monotreme) mammals. Imprinting has an important role in optimising pre-natal nutrition and growth, and most imprinted genes are expressed and imprinted in the placenta and developing fetus. In marsupials, however, the placental attachment is short-lived, and most growth and development occurs post-natally, supported by a changing milk composition tailor-made for each stage of development. Therefore there is a much greater demand on marsupial females during post-natal lactation than during pre-natal placentation, so there may be greater selection for genomic imprinting in the mammary gland than in the short-lived placenta. Recent studies in the tammar wallaby confirm the presence of genomic imprinting in nutrient-regulatory genes in the adult mammary gland. This suggests that imprinting may influence infant post-natal growth via the mammary gland as it does pre-natally via the placenta. Similarly, an increasing number of imprinted genes have been implicated in regulating feeding and nurturing behaviour in both the adult and the developing neonate/offspring in mice. Together these studies provide evidence that genomic imprinting is critical for regulating growth and subsequently the survival of offspring not only pre-natally but also post-natally.

GRB10 Imprinting Is Eutherian Mammal Specific

GRB10 is an imprinted gene differently expressed from two promoters in mouse and human. Mouse Grb10 is maternally expressed from the major promoter in most tissues and paternally expressed from the brain-specific promoter within specific regions of the fetal and adult central nervous system. Human GRB10 is biallelically expressed from the major promoter in most tissues except in the placental villus trophoblast where it is maternally expressed, whereas the brain-specific promoter is paternally expressed in the fetal brain. This study characterized the ortholog of GRB10 in a marsupial, the tammar wallaby (Macropus eugenii) to investigate the origin and evolution of imprinting at this locus. The protein coding exons and predicted amino acid sequence of tammar GRB10 were highly conserved with eutherian GRB10. The putative first exon, which is located in the orthologous region to the eutherian major promoter, was found in the tammar, but no exon was found in the downstream region corresponding to the eutherian brain-specific promoter, suggesting that marsupials only have a single promoter. Tammar GRB10 was widely expressed in various tissues including the brain but was not imprinted in any of the tissues examined. Thus, it is likely that GRB10 imprinting evolved in eutherians after the eutherian-marsupial divergence approximately 160 million years ago, subsequent to the acquisition of a brain-specific promoter, which resides within the imprinting control region in eutherians.

PRC2 is required for extensive reorganization of H3K27me3 during epigenetic reprogramming in mouse fetal germ cells

BackgroundDefining 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.ResultsHere, 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.ConclusionsCombined, 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.

WNT/β-catenin and p27/FOXL2 differentially regulate supporting cell proliferation in the developing ovary

Sexual development is initiated through differentiation of testicular Sertoli cells or ovarian granulosa cells. Although these supporting cells are considered to develop from common bipotential precursors, recent evidence suggests that distinct supporting cell populations are present in the ovary, with one providing granulosa cells of the medullary follicles and the other providing granulosa cells of the cortical follicles, the latter of which support lifelong fertility. Here, we demonstrate that XX fetal gonads contain GATA4 expressing supporting cells that either enter mitotic arrest, or remain proliferative. Blocking WNT signalling reduces XX supporting cell proliferation, while stabilising β-catenin signalling promotes proliferation, indicating that the renewal of pre-granulosa cells is dependent on WNT/β-catenin signalling in the proliferative supporting cell population. In contrast, XX supporting cells express p27 and FOXL2 and are maintained in mitotic arrest. Although FOXL2 is required for maintaining high levels of p27 expression, it is dispensable for entry and maintenance of mitotic arrest in XX supporting cells. Combined our data suggest that both medullary and cortical precursors arise from a common GATA4 expressing cell type. In addition, this work indicates that a balance between supporting cell self-renewal and differentiation is maintained in the developing ovary by relative WNT/β-catenin and p27/FOXL2 activities. This study provides significant new insights into the origin and formation of ovarian follicles and evidence supporting a common fetal origin of medullary and cortical granulosa cells.

FGF9, activin and TGFβ promote testicular characteristics in an XX gonad organ culture model

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 TGFβs, also present testis during testis formation. In this study, we investigated the potential of FGF9 combined with activin and TGFβ to induce testis development in cultured XX gonads. Our data demonstrated differing individual and combined abilities of FGF9, activin and TGFβ 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 TGFβ 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 β-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 TGFβ can induce testicular characteristics in XX gonads.

The importance of DNA repair for maintaining oocyte quality in response to anti-cancer treatments, environmental toxins and maternal ageing

BACKGROUND Within the ovary, oocytes are stored in long-lived structures called primordial follicles, each comprising a meiotically arrested oocyte, surrounded by somatic granulosa cells. It is essential that their genetic integrity is maintained throughout life to ensure that high quality oocytes are available for ovulation. Of all the possible types of DNA damage, DNA double-strand breaks (DSBs) are considered to be the most severe. Recent studies have shown that DNA DSBs can accumulate in oocytes in primordial follicles during reproductive ageing, and are readily induced by exogenous factors such as γ-irradiation, chemotherapy and environmental toxicants. DSBs can induce oocyte death or, alternatively, activate a program of DNA repair in order to restore genetic integrity and promote survival. The repair of DSBs has been intensively studied in the context of meiotic recombination, and in recent years more detail is becoming available regarding the repair capabilities of primordial follicle oocytes. OBJECTIVE AND RATIONALE This review discusses the induction and repair of DNA DSBs in primordial follicle oocytes. SEARCH METHODS PubMed (Medline) and Google Scholar searches were performed using the key words: primordial follicle oocyte, DNA repair, double-strand break, DNA damage, chemotherapy, radiotherapy, ageing, environmental toxicant. The literature was restricted to papers in the English language and limited to reports in animals and humans dated from 1964 until 2017. The references within these articles were also manually searched. OUTCOMES Recent experiments in animal models and humans have provided compelling evidence that primordial follicle oocytes can efficiently repair DNA DSBs arising from diverse origins, but this capacity may decline with increasing age. WIDER IMPLICATIONS Primordial follicle oocytes are vulnerable to DNA DSBs emanating from endogenous and exogenous sources. The ability to repair this damage is essential for female fertility. In the long term, augmenting DNA repair in primordial follicle oocytes has implications for the development of novel fertility preservation agents for female cancer patients and for the management of maternal ageing. However, further work is required to fully characterize the specific proteins involved and to develop strategies to bolster their activity.