Trichoplusia ni Transcriptomic Responses to the Phytosaponin Aglycone Hederagenin: Sex-Related Differences.

Many plant species, particularly legumes, protect themselves with saponins. Previously, a correlation was observed between levels of oleanolic acid-derived saponins, such as hederagenin-derived compounds, in the legume Medicago truncatula and caterpillar deterrence. Using concentrations that reflect the foliar levels of hederagenin-type saponins, the sapogenin hederagenin was not toxic to 4th instar caterpillars of the cabbage looper Trichoplusia ni nor did it act as a feeding deterrent. Female caterpillars consumed more diet than males, presumably to obtain the additional nutrients required for oogenesis, and are, thus, exposed to higher hederagenin levels. When fed the hederagenin diet, male caterpillars expressed genes encoding trypsin-like proteins (LOC113500509, LOC113501951, LOC113501953, LOC113501966, LOC113501965, LOC113499659, LOC113501950, LOC113501948, LOC113501957, LOC113501962, LOC113497819, LOC113501946, LOC113503910) as well as stress-responsive (LOC113503484, LOC113505107) proteins and cytochrome P450 6B2-like (LOC113493761) at higher levels than females. In comparison, female caterpillars expressed higher levels of cytochrome P450 6B7-like (LOC113492289). Bioinformatic tools predict that cytochrome P450s could catalyze the oxygenation of hederagenin which would increase the hydrophilicity of the compound. Expression of a Major Facilitator Subfamily (MFS) transporter (LOC113492899) showed a hederagenin dose-dependent increase in gene expression suggesting that this transporter may be involved in sapogenin efflux. These sex-related differences in feeding and detoxification should be taken into consideration in insecticide evaluations to minimize pesticide resistance.

Effect of sublethal concentrations of the bioinsecticide spinosyn treatment of Trichoplusia ni eggs on the caterpillar and its parasitoid, Trichogramma brassicae.

BACKGROUND: Integrated Pest Management (IPM) seeks to combine multiple management strategies for optimal pest control. One method that is successfully employed in IPM is the use of beneficial organisms. However, in severe circumstances when pest insects exceed threshold limits, insecticides may still need to be implemented. Thus, understanding the effects of insecticides on biocontrol agents, such as parasitoid wasps, is paramount to ensure sustainable agroecosystems. Sublethal effects of the bioinsecticide spinosyn, a mixture of the bacterial Saccharopolyspora spinosa (Mertz and Yao) fermentation products spinosyn A and D, on eggs of Trichoplusia ni (Hübner), a cruciferous crop pest, and its egg parasitoid Trichogramma brassicae (Bezdenko) was investigated. RESULTS: The LC50 for spinosyn A and D (dissolved in ethanol) on T. ni eggs is 54 ng mL-1 . Transcriptomics on caterpillars (1st and 3rd instars) that hatched from eggs treated with sublethal concentrations of spinosyn identified the upregulation of several genes encoding proteins that may be involved in insecticide resistance including detoxification enzymes, such as cytochrome P450 s, glutathione S-transferases and esterases. Sublethal T. ni egg treatments did not affect parasitoid emergence, however, there was a marked increase in the size of T. brassicae hind tibia and wings that emerged from spinosyn-treated eggs. CONCLUSIONS: For the caterpillar, treatment of eggs with sublethal concentrations of spinosyn may induce insecticide resistance mechanisms. For the parasitoids, their increased size when reared in spinosyn-treated eggs suggests that the emerged wasps may have higher performance. © 2024 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

The Association between Long-Term DDT or DDE Exposures and an Altered Sperm Epigenome-a Cross-Sectional Study of Greenlandic Inuit and South African VhaVenda Men.

BACKGROUND: The organochlorine dichlorodiphenyltrichloroethane (DDT) is banned worldwide owing to its negative health effects. It is exceptionally used as an insecticide for malaria control. Exposure occurs in regions where DDT is applied, as well as in the Arctic, where its endocrine disrupting metabolite, p,p'-dichlorodiphenyldichloroethylene (p,p'-DDE) accumulates in marine mammals and fish. DDT and p,p'-DDE exposures are linked to birth defects, infertility, cancer, and neurodevelopmental delays. Of particular concern is the potential of DDT use to impact the health of generations to come via the heritable sperm epigenome. OBJECTIVES: The objective of this study was to assess the sperm epigenome in relation to p,p'-DDE serum levels between geographically diverse populations. METHODS: In the Limpopo Province of South Africa, we recruited 247 VhaVenda South African men and selected 50 paired blood serum and semen samples, and 47 Greenlandic Inuit blood and semen paired samples were selected from a total of 193 samples from the biobank of the INUENDO cohort, an EU Fifth Framework Programme Research and Development project. Sample selection was based on obtaining a range of p,p'-DDE serum levels (mean=870.734±134.030 ng/mL). We assessed the sperm epigenome in relation to serum p,p'-DDE levels using MethylC-Capture-sequencing (MCC-seq) and chromatin immunoprecipitation followed by sequencing (ChIP-seq). We identified genomic regions with altered DNA methylation (DNAme) and differential enrichment of histone H3 lysine 4 trimethylation (H3K4me3) in sperm. RESULTS: Differences in DNAme and H3K4me3 enrichment were identified at transposable elements and regulatory regions involved in fertility, disease, development, and neurofunction. A subset of regions with sperm DNAme and H3K4me3 that differed between exposure groups was predicted to persist in the preimplantation embryo and to be associated with embryonic gene expression. DISCUSSION: These findings suggest that DDT and p,p'-DDE exposure impacts the sperm epigenome in a dose-response-like manner and may negatively impact the health of future generations through epigenetic mechanisms. Confounding factors, such as other environmental exposures, genetic diversity, and selection bias, cannot be ruled out. https://doi.org/10.1289/EHP12013.

Sperm histone H3 lysine 4 tri-methylation serves as a metabolic sensor of paternal obesity and is associated with the inheritance of metabolic dysfunction.

OBJECTIVE: Parental environmental exposures can strongly influence descendant risks for adult disease. How paternal obesity changes the sperm chromatin leading to the acquisition of metabolic disease in offspring remains controversial and ill-defined. The objective of this study was to assess (1) whether obesity induced by a high-fat diet alters sperm histone methylation; (2) whether paternal obesity can induce metabolic disturbances across generations; (3) whether there could be cumulative damage to the sperm epigenome leading to enhanced metabolic dysfunction in descendants; and (4) whether obesity-sensitive regions associate with embryonic epigenetic and transcriptomic profiles. Using a genetic mouse model of epigenetic inheritance, we investigated the role of histone H3 lysine 4 methylation (H3K4me3) in the paternal transmission of metabolic dysfunction. This transgenic mouse overexpresses the histone demethylase enzyme KDM1A in the developing germline and has an altered sperm epigenome at the level of histone H3K4 methylation. We hypothesized that challenging transgenic sires with a high-fat diet would further erode the sperm epigenome and lead to enhanced metabolic disturbances in the next generations. METHODS: To assess whether paternal obesity can have inter- or transgenerational impacts, and if so to identify potential mechanisms of this non-genetic inheritance, we used wild-type C57BL/6NCrl and transgenic males with a pre-existing altered sperm epigenome. To induce obesity, sires were fed either a control or high-fat diet (10% or 60% kcal fat, respectively) for 10-12 weeks, then bred to wild-type C57BL/6NCrl females fed a regular diet. F1 and F2 descendants were characterized for metabolic phenotypes by examining the effects of paternal obesity by sex, on body weight, fat mass distribution, the liver transcriptome, intraperitoneal glucose, and insulin tolerance tests. To determine whether obesity altered the F0 sperm chromatin, native chromatin immunoprecipitation-sequencing targeting H3K4me3 was performed. To gain insight into mechanisms of paternal transmission, we compared our sperm H3K4me3 profiles with embryonic and placental chromatin states, histone modification, and gene expression profiles. RESULTS: Obesity-induced alterations in H3K4me3 occurred in genes implicated in metabolic, inflammatory, and developmental processes. These processes were associated with offspring metabolic dysfunction and corresponded to genes enriched for H3K4me3 in embryos and overlapped embryonic and placenta gene expression profiles. Transgenerational susceptibility to metabolic disease was only observed when obese F0 had a pre-existing modified sperm epigenome. This coincided with increased H3K4me3 alterations in sperm and more severe phenotypes affecting their offspring. CONCLUSIONS: Our data suggest sperm H3K4me3 might serve as a metabolic sensor that connects paternal diet with offspring phenotypes via the placenta. This non-DNA-based knowledge of inheritance has the potential to improve our understanding of how environment shapes heritability and may lead to novel routes for the prevention of disease. This study highlights the need to further study the connection between the sperm epigenome, placental development, and children's health. SUMMARY SENTENCE: Paternal obesity impacts sperm H3K4me3 and is associated with placenta, embryonic and metabolic outcomes in descendants.

ChIP-seq protocol for sperm cells and embryos to assess environmental impacts and epigenetic inheritance.

In the field of epigenetic inheritance, delineating molecular mechanisms implicated in the transfer of paternal environmental conditions to descendants has been elusive. This protocol details how to track sperm chromatin intergenerationally. We describe mouse model design to probe chromatin states in single mouse sperm and techniques to assess pre-implantation embryo chromatin and gene expression. We place emphasis on how to obtain high-quality and quantifiable data sets in sperm and embryos, as well as highlight the limitations of working with low input. For complete details on the use and execution of this protocol, please refer to Lismer et al. (2021).

Histone H3 lysine 4 trimethylation in sperm is transmitted to the embryo and associated with diet-induced phenotypes in the offspring.

A father's lifestyle impacts offspring health; yet, the underlying molecular mechanisms remain elusive. We hypothesized that a diet that changes methyl donor availability will alter the sperm and embryo epigenomes to impact embryonic gene expression and development. Here, we demonstrate that a folate-deficient (FD) diet alters histone H3 lysine 4 trimethylation (H3K4me3) in sperm at developmental genes and putative enhancers. A subset of H3K4me3 alterations in sperm are retained in the pre-implantation embryo and associated with deregulated embryonic gene expression. Using a genetic mouse model in which sires have pre-existing altered H3K4me2/3 in sperm, we show that a FD diet exacerbates alterations in sperm H3K4me3 and embryonic gene expression, leading to an increase in developmental defect severity. These findings imply that paternal H3K4me3 is transmitted to the embryo and influences gene expression and development. It further suggests that epigenetic errors can accumulate in sperm to worsen offspring developmental outcomes.

Sperm histone H3 lysine 4 trimethylation is altered in a genetic mouse model of transgenerational epigenetic inheritance.

Advancing the molecular knowledge surrounding fertility and inheritance has become critical given the halving of sperm counts in the last 40 years, and the rise in complex disease which cannot be explained by genetics alone. The connection between both these trends may lie in alterations to the sperm epigenome and occur through environmental exposures. Changes to the sperm epigenome are also associated with health risks across generations such as metabolic disorders and cancer. Thus, it is imperative to identify the epigenetic modifications that escape reprogramming during spermatogenesis and embryogenesis. Here, we aimed to identify the chromatin signature(s) involved in transgenerational phenotypes in our genetic mouse model of epigenetic inheritance that overexpresses the histone demethylase KDM1A in their germ cells. We used sperm-specific chromatin immunoprecipitation followed by in depth sequencing (ChIP-seq), and computational analysis to identify whether differential enrichment of histone H3 lysine 4 trimethylation (H3K4me3), and histone H3 lysine 27 trimethylation (H3K27me3) serve as mechanisms for transgenerational epigenetic inheritance through the paternal germline. Our analysis on the sperm of KDM1A transgenic males revealed specific changes in H3K4me3 enrichment that predominantly occurred independently from bivalent H3K4me3/H3K27me3 regions. Many regions with altered H3K4me3 enrichment in sperm were identified on the paternal allele of the pre-implantation embryo. These findings suggest that sperm H3K4me3 functions in the transmission of non-genetic phenotypes transgenerationally.

Torpor expression is associated with differential spermatogenesis in hibernating eastern chipmunks.

Hibernators suppress physiological processes when expressing torpor, yet little is known about the effects of torpor on male reproductive physiology. Studies of hibernating mammals suggest that deep torpor negatively impacts spermatogenesis and that transitions between torpor and euthermic arousals increase cellular oxidative stress, with potentially damaging effects on sperm. Here, we hypothesize that variation in torpor expression affects the reproductive readiness of hibernators by impacting their sperm production. To test this, we examined the relationship between torpor expression and spermatogenesis in captive eastern chipmunks (Tamias striatus). We determined torpor depth with temperature data loggers and assessed its relationship with spermatogenesis by examining spermatogenic progression, cell division, sperm counts, sperm maturity, and DNA damage. We show that deep hibernators (high levels of torpor) largely halted spermatogenesis in late hibernation in comparison with shallow hibernators (low levels of torpor), where ongoing spermatogenesis was observed. Despite these differences in spermatogenic state during hibernation, spermatogenic progression, sperm numbers, and maturity did not differ in spring, potentially reflecting similar degrees of reproductive readiness. Interestingly, shallow hibernators exhibited higher rates of DNA damage in spermatogenic cells during hibernation, with this trend reversing in spring. Our results thus indicate that once heterothermy is terminated, deep hibernators resume spermatogenesis but are characterized by higher rates of DNA damage in spermatogenic cells at the seasonal stage when spring mating commences. Therefore, our study confirmed posthibernation recovery of sperm production but also a potential impact of deep torpor expression during winter on DNA damage in spring.

The genomic distribution of histone H3K4me2 in spermatogonia is highly conserved in sperm†.

Environmental exposures can alter the long-term health and development of offspring. How this environmental information is transmitted via the germline remains unknown, but it is thought to involve epigenetic inheritance. We recently determined that genetic disruption of histone H3 di-methylation at lysine 4 (H3K4me2) in sperm alters gene expression in the embryo and negatively impacts development across generations. However, little is known regarding when in spermatogenesis H3K4me2 methylation is established, and whether specific regions bearing H3K4me2 resist the epigenome remodeling that occurs throughout spermatogenesis. Our objective was to determine what genomic regions bearing histone H3K4me2 in spermatogonia are also present in sperm. Methods: Using transgenic mice expressing Oct4-GFP, we isolated an enriched spermatogonia population and performed ChIP-seq for H3K4me2, followed by downstream bioinformatics analysis. Using our epigenomic data and existing datasets, we compared the genomic distribution of H3K4me2 between spermatogonia and sperm. We also assessed the expression level of genes enriched in H3K4me2 in spermatogenic cell types and at specific embryonic developmental time-points. We observed that many regions of the sperm epigenome bearing H3K4me2 are already present in spermatogonia, suggesting an early establishment of this histone mark in spermatogenesis. Subsets of genes with a high enrichment in H3K4me2 in sperm are strongly expressed in spermatogenesis and others are associated with high gene expression during embryo development. These findings suggest that if epimutations in H3K4me2 are induced in spermatogonia they have the possibility to persist throughout spermatogenesis and may influence fertility by altering gene expression in spermatogenesis and in the embryo.

Disruption of histone methylation in developing sperm impairs offspring health transgenerationally.

A father's lifetime experiences can be transmitted to his offspring to affect health and development. However, the mechanisms underlying paternal epigenetic transmission are unclear. Unlike in somatic cells, there are few nucleosomes in sperm, and their function in epigenetic inheritance is unknown. We generated transgenic mice in which overexpression of the histone H3 lysine 4 (H3K4) demethylase KDM1A (also known as LSD1) during spermatogenesis reduced H3K4 dimethylation in sperm. KDM1A overexpression in one generation severely impaired development and survivability of offspring. These defects persisted transgenerationally in the absence of KDM1A germline expression and were associated with altered RNA profiles in sperm and offspring. We show that epigenetic inheritance of aberrant development can be initiated by histone demethylase activity in developing sperm, without changes to DNA methylation at CpG-rich regions.

The histone demethylase KDM1A is essential for the maintenance and differentiation of spermatogonial stem cells and progenitors.

Little is known of the fundamental processes governed by epigenetic mechanisms in the supplier cells of spermatogenesis, the spermatogonial stem cells (SSCs). The histone H3 lysine demethylase KDM1A is expressed in spermatogonia. We hypothesized that KDM1A serves in transcriptional regulation of SSCs and fertility. Using a conditional deletion of Kdm1a [conditional knockout (cKO)] in mouse spermatogonia, we determined that Kdm1a is essential for spermatogenesis as adult cKO males completely lack germ cells. Analysis of postnatal testis development revealed that undifferentiated and differentiating spermatogonial populations form in Kdm1a-cKO animals, yet the majority fail to enter meiosis. Loss of germ cells in the cKO was rapid with none remaining by postnatal day (PND) 21. To gain insight into the mechanistic implications of Kdm1a ablation, we isolated PND 6 spermatogonia enriched for SSCs and analyzed their transcriptome by RNA sequencing. Loss of Kdm1a was associated with altered transcription of 1206 genes. Importantly, differentially expressed genes between control and Kdm1a-cKO animals included those that are essential for SSC and progenitor maintenance and spermatogonial differentiation. The complete loss of fertility and failure to establish spermatogenesis indicate that Kdm1a is a master controller of gene transcription in spermatogonia and is required for SSC and progenitor maintenance and differentiation.

Impaired fertility and FSH synthesis in gonadotrope-specific Foxl2 knockout mice.

Impairments in pituitary FSH synthesis or action cause infertility. However, causes of FSH dysregulation are poorly described, in part because of our incomplete understanding of mechanisms controlling FSH synthesis. Previously, we discovered a critical role for forkhead protein L2 (FOXL2) in activin-stimulated FSH ß-subunit (Fshb) transcription in immortalized cells in vitro. Here, we tested the hypothesis that FOXL2 is required for FSH synthesis in vivo. Using a Cre/lox approach, we selectively ablated Foxl2 in murine anterior pituitary gonadotrope cells. Conditional knockout (cKO) mice developed overtly normally but were subfertile in adulthood. Testis size and spermatogenesis were significantly impaired in cKO males. cKO females exhibited reduced ovarian weight and ovulated fewer oocytes in natural estrous cycles compared with controls. In contrast, ovaries of juvenile cKO females showed normal responses to exogenous gonadotropin stimulation. Both male and female cKO mice were FSH deficient, secondary to diminished pituitary Fshb mRNA production. Basal and activin-stimulated Fshb expression was similarly impaired in Foxl2 depleted primary pituitary cultures. Collectively, these data definitively establish FOXL2 as the first identified gonadotrope-restricted transcription factor required for selective FSH synthesis in vivo.