Ingrid Sadler-Riggleman

The Helix-Loop-Helix Inhibitor of Differentiation (ID) Proteins Induce Post-Mitotic Terminally Differentiated Sertoli Cells to Re-Enter the Cell Cycle and Proliferate

Abstract Prior to puberty the Sertoli cells undergo active cell proliferation, and at the onset of puberty they become a terminally differentiated postmitotic cell population that support spermatogenesis. The molecular mechanisms involved in the postmitotic block of pubertal and adult Sertoli cells are unknown. The four known helix-loop-helix ID proteins (i.e., Id1, Id2, Id3, and Id4) are considered dominant negative regulators of cellular differentiation pathways and act as positive regulators of cellular proliferation. ID proteins are expressed at low levels by postpubertal Sertoli cells and are transiently induced by serum. The hypothesis tested was that ID proteins can induce a terminally differentiated postmitotic Sertoli cell to reenter the cell cycle if they are constitutively expressed. To test this hypothesis, ID1 and ID2 were stably integrated and individually overexpressed in postmitotic rat Sertoli cells. Overexpression of ID1 or ID2 allowed postmitotic Sertoli cells to reenter the cell cycle and undergo mitosis. The cells continued to proliferate even after 300 cell doublings. The functional markers of Sertoli cell differentiation such as transferrin, inhibin alpha, Sert1, and androgen binding protein (ABP) continued to be expressed by the proliferating Sertoli cells, but at lower levels. FSH receptor expression was lost in the proliferating Sertoli cell-Id lines. Some Sertoli cell genes, such as cyclic protein 2 (cathepsin L) and Sry-related HMG box protein-11 (Sox11) increase in expression. At no stage of proliferation did the cells exhibit senescence. The expression profile as determined with a microarray protocol of the Sertoli cell-Id lines suggested an overall increase in cell cycle genes and a decrease in growth inhibitory genes. These results demonstrate that overexpression of ID1 and ID2 genes in a postmitotic, terminally differentiated cell type have the capacity to induce reentry into the cell cycle. The observations are discussed in regards to potential future applications in model systems of terminally differentiated cell types such as neurons or myocytes.

Regulation of granulosa and theca cell transcriptomes during ovarian antral follicle development.

Coordinated interactions between ovarian granulosa and theca cells are required for female endocrine function and fertility. To elucidate these interactions the regulation of the granulosa and theca cell transcriptomes during bovine antral follicle development were investigated. Granulosa cells and theca cells were isolated from small (<5 mm), medium (5–10 mm), and large (>10 mm) antral bovine follicles. A microarray analysis of 24,000 bovine genes revealed that granulosa cells and theca cells each had gene sets specific to small, medium and large follicle cells. Transcripts regulated (i.e., minimally changed 1.5‐fold) during antral follicle development for the granulosa cells involved 446 genes and for theca cells 248 genes. Only 28 regulated genes were common to both granulosa and theca cells. Regulated genes were functionally categorized with a focus on growth factors and cytokines expressed and regulated by the two cell types. Candidate regulatory growth factor proteins mediating both paracrine and autocrine cell–cell interactions include macrophage inflammatory protein (MIP1 beta), teratocarcinoma‐derived growth factor 1 (TDGF1), stromal derived growth factor 1 (SDF1; i.e., CXCL12), growth differentiation factor 8 (GDF8), glia maturation factor gamma (GMFG), osteopontin (SPP1), angiopoietin 4 (ANGPT4), and chemokine ligands (CCL 2, 3, 5, and 8). The current study examined granulosa cell and theca cell regulated genes associated with bovine antral follicle development and identified candidate growth factors potentially involved in the regulation of cell–cell interactions required for ovarian function. Mol. Reprod. Dev. 75: 1457–1472, 2008.

SRY Directly Regulates the Neurotrophin 3 Promoter During Male Sex Determination and Testis Development in Rats

Neurotrophin 3 (Ntf3) is expressed in Sertoli cells and acts as a chemo-attractant for cell migration from the mesonephros into the developing testis, a process critical to the early morphological events of testis cord formation. The male sex-determining gene Sry initiates the process of testicular development. Sox9 is a key regulator of male sex determination and is directly regulated by SRY. Information on other downstream target genes of SRY is limited. The current study demonstrates an interaction of SRY with the Ntf3 promoter both in vitro and in vivo. The Ntf3 promoter in both rat and mouse contains at least one putative SRY binding site in the −0.6 kb promoter region. In a luciferase reporter assay system, both SRY and SOX9 stimulated the Ntf3 promoter in vitro through an interaction with this SRY-binding motif. In an immunoprecipitation-based pull-down assay, recombinant SRY protein bound the Ntf3 promoter fragment containing an intact SRY binding site, whereas the same protein did not interact with the fragment containing a mutated SRY motif. Specific antibodies against SRY were used in a chromatin immunoprecipitation (ChIP) assay of embryonic testis and were found to precipitate the Ntf3 promoter region. The SRY ChIP assay confirmed the direct interaction between SRY and the Ntf3 promoter in vivo during male sex determination. Observations suggest that SRY physically interacts with the Ntf3 promoter during male sex determination to coordinate cell migration in the testis to form testis cords.

Basic helix-loop-helix transcription factor TCF21 is a downstream target of the male sex determining gene SRY.

The cascade of molecular events involved in mammalian sex determination has been shown to involve the SRY gene, but specific downstream events have eluded researchers for decades. The current study identifies one of the first direct downstream targets of the male sex determining factor SRY as the basic-helix-loop-helix (bHLH) transcription factor TCF21. SRY was found to bind to the Tcf21 promoter and activate gene expression. Mutagenesis of SRY/SOX9 response elements in the Tcf21 promoter eliminated the actions of SRY. SRY was found to directly associate with the Tcf21 promoter SRY/SOX9 response elements in vivo during fetal rat testis development. TCF21 was found to promote an in vitro sex reversal of embryonic ovarian cells to induce precursor Sertoli cell differentiation. TCF21 and SRY had similar effects on the in vitro sex reversal gonadal cell transcriptomes. Therefore, SRY acts directly on the Tcf21 promoter to in part initiate a cascade of events associated with Sertoli cell differentiation and embryonic testis development.

Mercury-induced epigenetic transgenerational inheritance of abnormal neurobehavior is correlated with sperm epimutations in zebrafish

Abstract Methylmercury (MeHg) is a ubiquitous environmental neurotoxicant, with human exposures predominantly resulting from fish consumption. Developmental exposure of zebrafish to MeHg is known to alter their neurobehavior. The current study investigated the direct exposure and transgenerational effects of MeHg, at tissue doses similar to those detected in exposed human populations, on sperm epimutations (i.e., differential DNA methylation regions [DMRs]) and neurobehavior (i.e., visual startle and spontaneous locomotion) in zebrafish, an established human health model. F0 generation embryos were exposed to MeHg (0, 1, 3, 10, 30, and 100 nM) for 24 hours ex vivo. F0 generation control and MeHg-exposed lineages were reared to adults and bred to yield the F1 generation, which was subsequently bred to the F2 generation. Direct exposure (F0 generation) and transgenerational actions (F2 generation) were then evaluated. Hyperactivity and visual deficit were observed in the unexposed descendants (F2 generation) of the MeHg-exposed lineage compared to control. An increase in F2 generation sperm epimutations was observed relative to the F0 generation. Investigation of the DMRs in the F2 generation MeHg-exposed lineage sperm revealed associated genes in the neuroactive ligand-receptor interaction and actin-cytoskeleton pathways being effected, which correlate to the observed neurobehavioral phenotypes. Developmental MeHg-induced epigenetic transgenerational inheritance of abnormal neurobehavior is correlated with sperm epimutations in F2 generation adult zebrafish. Therefore, mercury can promote the epigenetic transgenerational inheritance of disease in zebrafish, which significantly impacts its environmental health considerations in all species including humans.

SRY Induced TCF21 Genome-Wide Targets and Cascade of bHLH Factors During Sertoli Cell Differentiation and Male Sex Determination in Rats

ABSTRACT Male sex determination is initiated through the testis-determining factor SRY that promotes Sertoli cell differentiation and subsequent gonadal development. The basic helix-loop-helix (bHLH) gene Tcf21 was identified as one of the direct downstream targets of SRY. The current study was designed to identify the downstream targets of TCF21 and the potential cascade of bHLH genes that promote Sertoli cell differentiation. A modified ChIP-Chip comparative hybridization analysis identified 121 direct downstream binding targets for TCF21. The gene networks and cellular pathways potentially regulated by these TCF21 targets were identified. One of the main bHLH targets for TCF21 was the bHLH gene scleraxis (Scx). An embryonic ovarian gonadal cell culture was used to examine the functional role of Sry, Tcf21, and Scx to promote an in vitro sex reversal and induction of Sertoli cell differentiation. SRY and TCF21 were found to induce the initial stages of Sertoli cell differentiation, whereas SCX was found to induce the later stages of Sertoli cell differentiation associated with pubertal development using transferrin gene expression as a marker. Therefore, a cascade of SRY followed by TCF21 followed by SCX appears to promote, in part, Sertoli cell fate determination and subsequent differentiation. The current observations help elucidate the initial molecular events involved in the induction of Sertoli cell differentiation and testis development.

Atrazine induced epigenetic transgenerational inheritance of disease, lean phenotype and sperm epimutation pathology biomarkers

Ancestral environmental exposures to a variety of environmental toxicants and other factors have been shown to promote the epigenetic transgenerational inheritance of adult onset disease. The current study examined the potential transgenerational actions of the herbicide atrazine. Atrazine is one of the most commonly used herbicides in the agricultural industry, in particular with corn and soy crops. Outbred gestating female rats were transiently exposed to a vehicle control or atrazine. The F1 generation offspring were bred to generate the F2 generation and then the F2 generation bred to generate the F3 generation. The F1, F2 and F3 generation control and atrazine lineage rats were aged and various pathologies investigated. The male sperm were collected to investigate DNA methylation differences between the control and atrazine lineage sperm. The F1 generation offspring (directly exposed as a fetus) did not develop disease, but weighed less compared to controls. The F2 generation (grand-offspring) was found to have increased frequency of testis disease and mammary tumors in males and females, early onset puberty in males, and decreased body weight in females compared to controls. The transgenerational F3 generation rats were found to have increased frequency of testis disease, early onset puberty in females, behavioral alterations (motor hyperactivity) and a lean phenotype in males and females. The frequency of multiple diseases was significantly higher in the transgenerational F3 generation atrazine lineage males and females. The transgenerational transmission of disease requires germline (egg or sperm) epigenetic alterations. The sperm differential DNA methylation regions (DMRs), termed epimutations, induced by atrazine were identified in the F1, F2 and F3 generations. Gene associations with the DMRs were identified. For the transgenerational F3 generation sperm, unique sets of DMRs (epimutations) were found to be associated with the lean phenotype or testis disease. These DMRs provide potential biomarkers for transgenerational disease. The etiology of disease appears to be in part due to environmentally induced epigenetic transgenerational inheritance, and epigenetic biomarkers may facilitate the diagnosis of the ancestral exposure and disease susceptibility. Observations indicate that although atrazine does not promote disease in the directly exposed F1 generation, it does have the capacity to promote the epigenetic transgenerational inheritance of disease.

Alterations in sperm DNA methylation, non-coding RNA and histone retention associate with DDT-induced epigenetic transgenerational inheritance of disease

BackgroundEnvironmental toxicants such as DDT have been shown to induce the epigenetic transgenerational inheritance of disease (e.g., obesity) through the germline. The current study was designed to investigate the DDT-induced concurrent alterations of a number of different epigenetic processes including DNA methylation, non-coding RNA (ncRNA) and histone retention in sperm.MethodsGestating females were exposed transiently to DDT during fetal gonadal development, and then, the directly exposed F1 generation, the directly exposed germline F2 generation and the transgenerational F3 generation sperm were investigated.ResultsDNA methylation and ncRNA were altered in each generation sperm with the direct exposure F1 and F2 generations being predominantly distinct from the F3 generation epimutations. The piRNA and small tRNA were the most predominant classes of ncRNA altered. A highly conserved set of histone retention sites were found in the control lineage generations which was not significantly altered between generations, but a large number of new histone retention sites were found only in the transgenerational generation DDT lineage sperm.ConclusionsTherefore, all three different epigenetic processes were concurrently altered as DDT induced the epigenetic transgenerational inheritance of sperm epimutations. The direct exposure generations sperm epigenetic alterations were distinct from the transgenerational sperm epimutations. The genomic features and gene associations with the epimutations were investigated to help elucidate the integration of these different epigenetic processes. Observations demonstrate all three epigenetic processes are involved in transgenerational inheritance. The different epigenetic processes appear to be integrated in mediating the epigenetic transgenerational inheritance phenomenon.

Differential DNA Methylation Regions in Adult Human Sperm following Adolescent Chemotherapy: Potential for Epigenetic Inheritance

Background The potential that adolescent chemotherapy can impact the epigenetic programming of the germ line to influence later life adult fertility and promote epigenetic inheritance was investigated. Previous studies have demonstrated a number of environmental exposures such as abnormal nutrition and toxicants can promote sperm epigenetic changes that impact offspring. Methods Adult males approximately ten years after pubertal exposure to chemotherapy were compared to adult males with no previous exposure. Sperm were collected to examine differential DNA methylation regions (DMRs) between the exposed and control populations. Gene associations and correlations to genetic mutations (copy number variation) were also investigated. Methods and Findings A signature of statistically significant DMRs was identified in the chemotherapy exposed male sperm. The DMRs, termed epimutations, were found in CpG desert regions of primarily 1 kilobase size. Observations indicate adolescent chemotherapy exposure can promote epigenetic alterations that persist in later life. Conclusions This is the first observation in humans that an early life chemical exposure can permanently reprogram the spermatogenic stem cell epigenome. The germline (i.e., sperm) epimutations identified suggest chemotherapy has the potential to promote epigenetic inheritance to the next generation.

Epigenetic Transgenerational Inheritance of Altered Sperm Histone Retention Sites

A variety of environmental toxicants and factors have been shown to induce the epigenetic transgenerational inheritance of disease and phenotypic variation. Epigenetic alterations in the germline (sperm or egg) are required to transmit transgenerational phenotypes. The current study was designed to investigate the potential role of histones in sperm to help mediate the epigenetic transgenerational inheritance. The agricultural fungicide vinclozolin and the pesticide DDT (dichlorodiphenyltrichloroethane) were independently used to promote the epigenetic transgenerational inheritance of disease. Purified cauda epididymal sperm were collected from the transgenerational F3 generation control and exposure lineage male rats for histone analysis. A reproducible core of histone H3 retention sites was observed using an H3 chromatin immunoprecipitation (ChIP-Seq) analysis in control lineage sperm. Interestingly, the same core group of H3 retention sites plus additional differential histone retention sites (DHRs) were observed in the F3 generation exposure lineage sperm. Although new histone H3 retention sites were observed, negligible change in histone modification (methylation of H3K27me3) was observed between the control and exposure lineages. Observations demonstrate that in addition to alterations in sperm DNA methylation and ncRNA previously identified, the induction of differential histone retention sites (DHRs) also appear to be involved in environmentally induced epigenetic transgenerational inheritance.