DOT1-like (DOT1L) histone methyltransferase is essential for mammalian erythropoiesis. Loss of DOT1L in knockout (Dot1l-KO) mouse embryos resulted in lethal anemia at midgestational age. The only recognized molecular function of DOT1L is its methylation of histone H3 lysine 79 (H3K79). We generated a Dot1l methyltransferase mutant (Dot1l-MM) mouse model to determine the role of DOT1L methyltransferase activity in early embryonic hematopoiesis. Dot1l-MM embryos failed to survive beyond embryonic day 13.5 (E13.5), similarly to Dot1l-KO mice. However, when examined at E10.5, Dot1l-MM embryos did not exhibit overt anemia like the Dot1l-KO. Vascularity and the presence of red blood cells in the Dot1l-MM yolk sacs as well as in the AGM region of Dot1l-MM embryos appeared to be similar to that of wildtype. In ex vivo cultures of yolk sac cells, Dot1l-MM primitive erythroblasts formed colonies comparable to those of the wildtype. Although ex vivo cultures of Dot1l-MM definitive erythroblasts formed relatively smaller colonies, inhibition of DOT1L methyltransferase activity in vivo by administration of EPZ-5676 minimally affected the erythropoiesis. Our results indicate that early embryonic erythropoiesis in mammals requires a DOT1L function that is independent of its intrinsic methyltransferase activity.

Recent studies documented that the selective estrogen receptor modulator tamoxifen prevents follicle loss and promotes fertility following in vivo exposure of rodents to irradiation or ovotoxic cancer drugs, cyclophosphamide and doxorubicin. In an effort to characterize the ovarian-sparing mechanisms of tamoxifen in preantral follicle classes, cultured neonatal rat ovaries (Day 4, Sprague Dawley) were treated for 1-7 days with active metabolites of cyclophosphamide (i.e., 4-hydroxycyclophosphamide; CTX) (0, 1, and 10 μM) and tamoxifen (i.e., 4-hydroxytamoxifen; TAM) (0 and 10 μM) in vitro, and both apoptosis and follicle numbers were measured. CTX caused marked follicular apoptosis and follicular loss. TAM treatment decreased follicular loss and apoptosis from CTX in vitro. TAM alone had no effect on these parameters. IGF-1 and IGF-1 receptor were assessed in ovarian tissue showing no impact of TAM or CTX on these endpoints. Targeted mRNA analysis during follicular rescue by TAM revealed decreased expression of multiple genes related to inflammation, including mediators of lipoxygenase and prostaglandin production and signaling (Alox5, Pla2g1b, Ptgfr), cytokine binding (Il1r1, Il2rg ), apoptosis (Tnfrsf1a), second messenger signaling (Mapk1, Mapk14, Plcg1), as well as tissue remodeling and vasodilation (Bdkrb2, Klk15). The results suggest that TAM protects the ovary from CTX-mediated toxicity through direct ovarian actions that oppose follicular loss.

The compound 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a by-product of human industrial activity, was found to affect ovarian steroidogenesis in animals, but the mechanism of its action is still unclear. The aims of the study were to examine the effect of TCDD on (1) progesterone (P 4 ) and oestradiol (E 2 ) production by granulosa cells isolated from medium (3-6 mm) and preovulatory (≥ 8 mm) porcine follicles, (2) the viability of the cells, and (3) the incidence of apoptosis. Porcine granulosa cells were cultured (48 h) with or without TCDD (100 pM, 100 nM). Steroid hormone concentrations in the medium were determined by radioimmunoassay. The viability of granulosa cells was tested spectrophotometrically (alamarBlue™ assay). Apoptosis was evaluated by flow cytometry using Annexin V and by TUNEL assay. The higher dose of TCDD (100 nM) significantly inhibited P 4 and stimulated E 2 production by luteinised granulosa cells isolated from medium follicles. The lower dose of TCDD (100 pM) significantly stimulated P 4 and inhibited E 2 secretion by the cells isolated from preovulatory follicles. None of the two TCDD doses affected cell viability or induced apoptosis in granulosa cells. In conclusion, TCDD directly affected steroid production by granulosa cells obtained from mature pigs, but the effect of TCDD was not due to its cytotoxicity.

Low doses of endocrine disrupting chemicals (EDCs) used in combination may act in a manner different from that of individual compounds. The objective of the study was to examine in vitro effects of low doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 100 pM) and genistein (500 nM) on: 1) progesterone (P4) and estradiol (E2) secretion (48 h); 2) dynamic changes in aryl hydrocarbon receptor (AhR) mRNA and protein expression (1, 3, 6, 24 and 48 h); 3) dynamic changes in estrogen receptor β (ERβ) mRNA and protein expression (1, 3, 6, 24 and 48 h); and 4) induction of apoptosis in porcine granulosa cells derived from medium follicles (3, 6 and 24 h). TCDD had no effect on P4 or E2 production, but potentiated the inhibitory effect of genistein on P4 production. In contrast to the individual treatments which did not produce any effects, TCDD and genistein administered together decreased ERβ and AhR protein expression in granulosa cells. Moreover, the inhibitory effect of TCDD on AhR mRNA expression was abolished by genistein. The treatments did not induce apoptosis in the cells. In summary, combined effects of low concentrations of TCDD and genistein on follicular function of pigs differed from that of individual compounds. The results presented in the current paper clearly indicate that effects exerted by low doses of EDCs applied in combination must be taken into consideration when studying potential risk effects of EDCs on biological processes.

No abstract

Disruptor of Telomere silencing 1-Like (DOT1L), is a histone 3, lysine 79 (H3K79) methyltransferase that has been implicated in multiple processes, including activation of transcription, regulation of the cell cycle, leukemogenesis, and mouse embryonic development. In previous studies, we found that Dot1L deficiency results in an erythropoietic defect, leading to lethal anemia at around mid-gestation (Feng et al., 2010, Blood). The precise molecular mechanism(s) by which DOT1L regulates embryonic hematopoiesis has not yet been elucidated and is the overall objective of this study. DOT1L is a large protein (1540aa), and it is involved in several, diverse processes. However, its only documented activity has been as an intrinsic, histone methyltransferase. Additional functional domains of the protein might be responsible for its diverse activities, including murine hematopoiesis. We sought to determine whether the methyltransferase activity of DOT1L is essential for hematopoiesis. To test this hypothesis, we developed a Dot1L methyltransferase mutant (Dot1L MM) mouse line. Using the Cas9/CRISPR system, we created a Dot1L point mutation in cultured murine embryonic stem cells (mESC). These mESCs contained a wildtype allele, and the second had a single amino acid change in the methyltransferase domain of Dot1L, thereby eliminating its methyltransferase activity, but preserving the rest of the protein. We injected these mutant mESCs into blastocysts to produce chimeric mice. The chimeras containing the methyltransferase mutation were back crossed onto the C57/BL6 background, producing male and female offspring heterozygous for the mutation. Through intercrosses of the F1 generation, we found that the Dot1L MM mice displayed an embryonic lethality between embryonic days 10.5 and 13.5, similar to the Dot1L knockout mice, as reported in our previous studies. We additionally performed ex vivo blood differentiation assays and extensively self-renewing erythroblast (ESRE) cultures using E10.5 yolk sacs from Dot1L MM and knockout mice. Our data showed that the Dot1L MM and knockout yolk sacs display similar phenotypes. In blood differentiation cultures, Dot1L knockout and MM yolk sac cells form the same types of hematopoietic colonies as in wildtype (both erythroid and myeloid), but there is a decrease in colony size and number compared to the wildtype. In the ESRE cultures, Dot1L knockout and MM yolk sac cells form significantly fewer ESREs and have increased cell death compared to wildtype. Strikingly, the cells in these cultures also exhibit a profound genomic instability, implicating DOT1L methyltransferase activity in maintenance of the genome as well as the viability of hematopoietic progenitors. These results suggest that the methyltransferase activity of DOT1L plays a predominant role in the activity of the protein as a whole, and is responsible for its function in facilitating early murine hematopoiesis. Disclosures No relevant conflicts of interest to declare.