Anti-Müllerian hormone and inhibin-B concentrations vary cyclically in nonovulating queens within reference ranges established for determining gonadal status in cats.

OBJECTIVE: To define cyclic changes in anti-Müllerian hormone (AMH), inhibin-B, and progesterone concentrations and establish statistically valid, population-based clinical reference ranges in queens. ANIMALS: Cyclic queens (fertile, n = 6; infertile, 6) from an institutional breeding colony were blood sampled longitudinally, each for over 2 months, between November 2021 and February 2022, and residual serum samples from intact (n = 205) and ovariohysterectomized (49) queens from clinical submissions were used to establish reference ranges for intact and spayed females. METHODS: AMH and inhibin-B were measured using commercially available ELISAs, progesterone was measured using an in-house ELISA, and 90% CIs were calculated from these data. RESULTS: AMH and inhibin-B fluctuated in a highly correlated, cyclic pattern in 3 queens that did not ovulate immediately, whereas AMH declined as progesterone increased, indicative of ovulation, which occurred spontaneously early in the sampling period in 3 others; statistically valid reference ranges were established in intact and ovariohysterectomized females. CLINICAL RELEVANCE: Cyclic changes in hormone profiles were defined, providing relevant context for interpreting results in cases seeking to determine gonadal status (presence or absence of gonadal tissue) on the basis of established, population-based reference ranges reported here for cats for the first time.

myc maintains embryonic stem cell pluripotency and self-renewal.

While endogenous Myc (c-myc) and Mycn (N-myc) have been reported to be separately dispensable for murine embryonic stem cell (mESC) function, myc greatly enhances induced pluripotent stem (iPS) cell formation and overexpressed c-myc confers LIF-independence upon mESC. To address the role of myc genes in ESC and in pluripotency generally, we conditionally knocked out both c- and N-myc using myc doubly homozygously floxed mESC lines (cDKO). Both lines of myc cDKO mESC exhibited severely disrupted self-renewal, pluripotency, and survival along with enhanced differentiation. Chimeric embryos injected with DKO mESC most often completely failed to develop or in rare cases survived but with severe defects. The essential nature of myc for self-renewal and pluripotency is at least in part mediated through orchestrating pluripotency-related cell cycle and metabolic programs. This study demonstrates that endogenous myc genes are essential for mESC pluripotency and self-renewal as well as providing the first evidence that myc genes are required for early embryogenesis, suggesting potential mechanisms of myc contribution to iPS cell formation.

Endogenous mammalian histone H3.3 exhibits chromatin-related functions during development.

BACKGROUND: The histone variant H3.3 plays key roles in regulating chromatin states and transcription. However, the role of endogenous H3.3 in mammalian cells and during development has been less thoroughly investigated. To address this gap, we report the production and phenotypic analysis of mice and cells with targeted disruption of the H3.3-encoding gene, H3f3b. RESULTS: H3f3b knockout (KO) mice exhibit a semilethal phenotype traceable at least in part to defective cell division and chromosome segregation. H3f3b KO cells have widespread ectopic CENP-A protein localization suggesting one possible mechanism for defective chromosome segregation. KO cells have abnormal karyotypes and cell cycle profiles as well. The transcriptome and euchromatin-related epigenome were moderately affected by loss of H3f3b in mouse embryonic fibroblasts (MEFs) with ontology most notably pointing to changes in chromatin regulatory and histone coding genes. Reduced numbers of H3f3b KO mice survive to maturity and almost all survivors from both sexes are infertile. CONCLUSIONS: Taken together, our studies suggest that endogenous mammalian histone H3.3 has important roles in regulating chromatin and chromosome functions that in turn are important for cell division, genome integrity, and development.

Methyl deficiency causes reduction of the methyl-CpG-binding protein, MeCP2, in rat liver.

MeCP2 is a member of a family of proteins [methyl- (cytosine-guanine)CpG-binding proteins] that bind specifically to methylated DNA and induce chromatin remodeling and gene silencing. Dietary deficiency of folate, choline and methionine causes decreased tissue S-adenosylmethionine concentrations (methyl deficiency), global DNA hypomethylation, hepatic steatosis, cirrhosis and ultimately hepatic tumorigenesis in rodents. We investigated the effects of this diet on expression of MeCP2 during pre-neoplastic transformation of liver tissue. After 9 weeks, MeCP2 mRNA level was slightly higher in methyl-deficient rats compared with replete controls, while after 36 weeks, a difference in MeCP2 mRNA level was no longer observed. In contrast, MeCP2 protein level was reduced almost 2-fold in the deficient rats compared with replete controls at both 9 and 36 weeks. Conversely, a second methyl-CpG-binding protein, MBD2, showed increased levels of both message and protein at the two time points. Low MeCP2 protein in the deficient rats was associated with a low level of the co-repressor protein, Sin3a, at 36 weeks. Moreover, a known gene target of MeCP2, the tumor suppressor gene metallothionein-I, was over-expressed in the deficient rat livers at both 9 and 36 weeks, suggesting that reduction in MeCP2 may have functional consequences. Methyl deficiency also caused an increase in the ratio of long to short variants of MeCP2 transcripts. This finding suggests that reduced MeCP2 protein level is the result of a reduced rate of translation. Reduction of MeCP2 protein expression may influence the initiation and/or progression of hepatic cancer induced by methyl deficiency and may provide a useful marker of pre-neoplastic change.