The embryonic genome is formed by fusion of a maternal and a paternal genome. To accommodate the resulting diploid genome in the fertilized oocyte dramatic global genome reorganizations must occur. The higher order structure of chromatin in vivo is critically dependent on architectural chromatin proteins, with the family of linker histone proteins among the most critical structural determinants. Although somatic cells contain numerous linker histone variants, only one, H1FOO, is present in mouse oocytes. Upon fertilization H1FOO rapidly populates the introduced paternal genome and replaces sperm-specific histone-like proteins. The same dynamic replacement occurs upon introduction of a nucleus during somatic cell nuclear transfer. To understand the molecular basis of this dynamic histone replacement process, we compared the localization and binding dynamics of somatic H1 and oocyte-specific H1FOO and identified the molecular determinants of binding to either oocyte or somatic chromatin in living cells. We find that although both histones associate readily with chromatin in nuclei of somatic cells, only H1FOO is capable of correct chromatin association in the germinal vesicle stage oocyte nuclei. This specificity is generated by the N-terminal and globular domains of H1FOO. Measurement of in vivo binding properties of the H1 variants suggest that H1FOO binds chromatin more tightly than somatic linker histones. We provide evidence that both the binding properties of linker histones as well as additional, active processes contribute to the replacement of somatic histones with H1FOO during nuclear transfer. These results provide the first mechanistic insights into the crucial step of linker histone replacement as it occurs during fertilization and somatic cell nuclear transfer.

We previously reported the discovery of a novel mammalian H1 linker histone termed H1FOO (formerly H1OO), a replacement H1, the expression of which is restricted to the growing/ maturing oocyte and to the zygote. The significance of this pre-embryonic H1 draws on its substantial orthologous conservation, singular structural attributes, selectivity for the germ cell lineage, prolonged nucleosomal residence, and apparent predominance among germ cell H1s. Herein, we report that the intronic, single-copy, five-exon (> or =5301 base pair) H1foo gene maps to chromosome 6 and that the corresponding primary H1foo transcript gives rise to two distinct, alternatively spliced mRNA species (H1foo(alpha) and H1foo(beta)). The expression of the oocytic H1FOO transcript and protein proved temporally coupled to the recruitment of resting primordial follicles into a developing primary follicular cohort and thus to the critical transition marking the onset of oocytic growth. The corresponding potential protein isoforms (H1FOO(alpha) and H1FOO(beta)), both nuclear localization sequence-endowed but export consensus sequence-free and possessing a significant net positive charge, localized primarily to perinucleolar heterochromatin in the oocytic germinal vesicle. Further investigation will be required to define the functional role of the H1FOO protein in the ordering of the chromatin of early mammalian development as well as its potential role in defining the primordial-to-primary follicle transition.

The success of TYS will depend on the acceptance of TYS by all the relevant pathology and gynecologic oncology communities who, by their joint efforts, will adopt, critically evaluate, and optimize this method with the only aim of further improving the impact of endometrial cytology on patients' care.

The relationship between alterations in gene expression and differences in developmental potential in primate oocytes and embryos was examined. Oocytes from 3 sources were used for these studies: 1) in vivo-matured oocytes from monkeys stimulated with FSH and hCG, 2) in vitro-matured oocytes from large follicles of monkeys primed with FSH, and 3) in vitro-matured oocytes from small follicles from nonstimulated (NS) monkeys. Following in vitro fertilization, embryos from these oocytes displayed high, moderate, and low developmental competence, respectively. Oocytes from NS females displayed aberrant accumulation of a number of maternal mRNAs, followed by precocious loss of many maternal mRNAs by the 2-cell stage. Embryos from NS oocytes displayed alterations in expression of key transcription factors after the 8-cell stage. Oocytes and embryos from FSH-stimulated females also displayed alterations in gene expression relative to hCG-stimulated females, but these alterations were much less severe than those observed for NS oocytes and embryos. Our data are consistent with the hypothesis that continued development and maturation of the oocyte within the ovarian follicle in vivo facilitates the production of oocytes of the highest developmental potential, and that in vitro conditions may not support this process as effectively due to differences in the extracellular milieu. These observations are relevant to understanding the role of the in vivo environment on oocyte maturation, and the potential effects of in vitro maturation on human assisted reproduction methods.