Transcriptomic analysis of the seminal vesicle response to the reproductive toxicant acrylamide

Background: The seminal vesicles synthesise bioactive factors that support gamete function, modulate the female reproductive tract to promote implantation, and influence developmental programming of offspring phenotype. Despite the significance of the seminal vesicles in reproduction, their biology remains poorly defined. Here, to advance understanding of seminal vesicle biology, we analyse the mouse seminal vesicle transcriptome under normal physiological conditions and in response to acute exposure to the reproductive toxicant acrylamide. Mice were administered acrylamide (25 mg/kg bw/day) or vehicle control daily for five consecutive days prior to collecting seminal vesicle tissue 72 h following the final injection. Results: A total of 15,304 genes were identified in the seminal vesicles with those encoding secreted proteins amongst the most abundant. In addition to reproductive hormone pathways, functional annotation of the seminal vesicle transcriptome identified cell proliferation, protein synthesis, and cellular death and survival pathways as prominent biological processes. Administration of acrylamide elicited 70 differentially regulated (fold-change ≥1.5 or ≤ 0.67) genes, several of which were orthogonally validated using quantitative PCR. Pathways that initiate gene and protein synthesis to promote cellular survival were prominent amongst the dysregulated pathways. Inflammation was also a key transcriptomic response to acrylamide, with the cytokine, Colony stimulating factor 2 (Csf2) identified as a top-ranked upstream driver and inflammatory mediator associated with recovery of homeostasis. Early growth response (Egr1), C-C motif chemokine ligand 8 (Ccl8), and Collagen, type V, alpha 1 (Col5a1) were also identified amongst the dysregulated genes. Additionally, acrylamide treatment led to subtle changes in the expression of genes that encode proteins secreted by the seminal vesicle, including the complement regulator, Complement factor b (Cfb). Conclusions: These data add to emerging evidence demonstrating that the seminal vesicles, like other male reproductive tract tissues, are sensitive to environmental insults, and respond in a manner with potential to exert impact on fetal development and later offspring health.David A. Skerrett-Byrne, Brett Nixon, Elizabeth G. Bromfield, James Breen, Natalie A. Trigg, Simone J. Stanger, Ilana R. Bernstein, Amanda L. Anderson, Tessa Lord, R. John Aitken, Shaun D. Roman, Sarah A. Robertson, and John E. Schjenke

Proteomic dissection of the impact of environmental exposures on mouse seminal vesicle function

Seminal vesicles are an integral part of the male reproductive accessory gland system. They produce a complex array of secretions containing bioactive constituents that support gamete function and promote reproductive success, with emerging evidence suggesting these secretions are influenced by our environment. Despite their significance, the biology of seminal vesicles remains poorly defined. Here, we complete the first proteomic assessment of mouse seminal vesicles and assess the impact of the reproductive toxicant acrylamide. Mice were administered acrylamide (25 mg/kg bw/day) or control daily for five consecutive days prior to collecting seminal vesicle tissue. A total of 5,013 proteins were identified in the seminal vesicle proteome with bioinformatic analyses identifying cell proliferation, protein synthesis, cellular death and survival pathways as prominent biological processes. Secreted proteins were amongst the most abundant and several proteins are linked with seminal vesicle phenotypes. Analysis of the effect of acrylamide on the seminal vesicle proteome revealed 311 differentially regulated (FC ± 1.5, p ≤ 0.05, 205 up-regulated, 106 down-regulated) proteins, orthogonally validated via immunoblotting and immunohistochemistry. Pathways that initiate protein synthesis to promote cellular survival were prominent amongst the dysregulated pathways and Rapamycin-insensitive companion of mTOR (RICTOR, p = 6.69E-07) was a top-ranked upstream driver. Oxidative stress was implicated as contributing to protein changes, with acrylamide causing an increase in 8-OHdG in seminal vesicle epithelial cells (5-fold increase, p = 0.016) and the surrounding smooth muscle layer (2-fold increase, p = 0.043). Additionally, acrylamide treatment caused a reduction in seminal vesicle secretion weight (36% reduction, p = 0.009) and total protein content (25% reduction, p = 0.017). Together these findings support the interpretation that toxicant exposure influences male accessory gland physiology and highlights the need to consider the response of all male reproductive tract tissues when interpreting the impact of environmental stressors on male reproductive function.David A. Skerrett-Byrne, Natalie A. Trigg, Elizabeth G. Bromfield, Matthew D. Dun, Ilana R. Bernstein, Amanda L. Anderson ... et al

Induction and selection of Sox17-expressing endoderm cells generated from murine embryonic stem cells

Embryonic stem (ES) cells offer a valuable source for generating insulin-producing cells. However, current differentiation protocols often result in heterogeneous cell populations of various developmental stages. Here we show the activin A-induced differentiation of mouse ES cells carrying a homologous dsRed-IRES-puromycin knock-in within the Sox17 locus into the endoderm lineage. Sox17-expressing cells were selected by fluorescence-assisted cell sorting (FACS) and characterized at the transcript and protein level. Treatment of ES cells with high concentrations of activin A for 10 days resulted in up to 19% Sox17-positive cells selected by FACS. Isolated Sox17-positive cells were characterized by defini- tive endoderm-specific Sox17/Cxcr4/Foxa2 transcripts, but lacked pluripotency-associated Oct4 mRNA and protein. The Sox17-expressing cells showed downregulation of extraembryonic endoderm (Sox7, Afp, Sdf1)-, mesoderm (Foxf1, Meox1)- and ectoderm (Pax6, NeuroD6)-specific transcripts. The presence of Hnf4alpha, Hes1 and Pdx1 mRNA demonstrated the expression of primitive gut/foregut cell-specific markers. Ngn3, Nkx6.1 and Nkx2.2 transcripts in Sox17-positive cells were determined as properties of pancreatic endocrine progenitors. Immunocytochemistry of activin A-induced Sox17-positive embryoid bodies revealed coexpression of Cxcr4 and Foxa2. Moreover, the histochemical demonstration of E-cadherin-, Cxcr4-, Sox9-, Hnf1beta- and Ngn3-positive epithelial-like structures underlined the potential of Sox17-positive cells to further differentiate into the pancreatic lineage. By reducing the heterogeneity of the ES cell progeny, Sox17-expressing cells are a suitable model to evaluate the effects of growth and differentiation factors and of culture conditions to delineate the differentiation process for the generation of pancreatic cells in vitro