Loss of heterochromatin and retrotransposon silencing as determinants in oocyte aging.

Mammalian oocyte quality reduces with age. We show that prior to the occurrence of significant aneuploidy (9M in mouse), heterochromatin histone marks are lost, and oocyte maturation is impaired. This loss occurs in both constitutive and facultative heterochromatin marks but not in euchromatic active marks. We show that heterochromatin loss with age also occurs in human prophase I-arrested oocytes. Moreover, heterochromatin loss is accompanied in mouse oocytes by an increase in RNA processing and associated with an elevation in L1 and IAP retrotransposon expression and in DNA damage and DNA repair proteins nuclear localization. Artificial inhibition of the heterochromatin machinery in young oocytes causes an elevation in retrotransposon expression and oocyte maturation defects. Inhibiting retrotransposon reverse-transcriptase through azidothymidine (AZT) treatment in older oocytes partially rescues their maturation defects and activity of the DNA repair machinery. Moreover, activating the heterochromatin machinery via treatment with the SIRT1 activating molecule SRT-1720, or overexpression of Sirt1 or Ezh2 via plasmid electroporation into older oocytes causes an upregulation in constitutive heterochromatin, downregulation of retrotransposon expression, and elevated maturation rates. Collectively, our work demonstrates a significant process in oocyte aging, characterized by the loss of heterochromatin-associated chromatin marks and activation of specific retrotransposons, which cause DNA damage and impair oocyte maturation.

Pre-pubertal oocytes harbor altered histone modifications and chromatin configuration.

Pre-pubertal oocytes are still dormant. They are arrested in a GV state and do not undergo meiotic divisions naturally. A multitude of molecular pathways are changed and triggered upon initiation of puberty. It is not yet clear which epigenetic events occur in oocytes upon pubertal transition, and how significant these epigenetic events may be. We evaluated epigenetic marker levels in mouse pre-pubertal and post-pubertal female oocytes. In addition, we evaluated H3K9me2 levels in human oocytes collected from fertility preservation patients, comparing the levels between pre-pubertal patients and post-pubertal patients. The chromatin structure shows a lower number of chromocenters in mouse post-pubertal oocytes in comparison to pre-pubertal oocytes. All heterochromatin marker levels checked (H3K9me2, H3K27me3, H4K20me1) significantly rise across the pubertal transition. Euchromatin markers vary in their behavior. While H3K4me3 levels rise with the pubertal transition, H3K27Ac levels decrease with the pubertal transition. Treatment with SRT1720 [histone deacetylase (HDAC) activator] or overexpression of heterochromatin factors does not lead to increased heterochromatin in pre-pubertal oocytes. However, treatment of pre-pubertal oocytes with follicle-stimulating hormone (FSH) for 24 h - changes their chromatin structure to a post-pubertal configuration, lowers the number of chromocenters and elevates their histone methylation levels, showing that hormones play a key role in chromatin regulation of pubertal transition. Our work shows that pubertal transition leads to reorganization of oocyte chromatin and elevation of histone methylation levels, thus advancing oocyte developmental phenotype. These results provide the basis for finding conditions for in-vitro maturation of pre-pubertal oocytes, mainly needed to artificially mature oocytes of young cancer survivors for fertility preservation purposes.

Age-Dependent in vitro Maturation Efficacy of Human Oocytes - Is There an Optimal Age?

In vitro maturation of oocytes from antral follicles seen during tissue harvesting is a fertility preservation technique with potential advantages over ovarian tissue cryopreservation (OTC), as mature frozen and later thawed oocyte used for fertilization poses decreased risk of malignant cells re-seeding, as compared to ovarian tissue implantation. We previously demonstrated that in vitro maturation (IVM) performed following OTC in fertility preservation patients, even in pre-menarche girls, yields a fair amount of oocytes available for IVM and freezing for future use. We conducted a retrospective cohort study, evaluating IVM outcomes in chemotherapy naïve patients referred for fertility preservation by OTC that had oocyte collected from the medium with attempted IVM. A total of 133 chemotherapy naïve patients aged 1-35 years were included in the study. The primary outcome was IVM rate in the different age groups - pre-menarche (1-5 and ≥6 years), post-menarche (menarche-17 years), young adults (18-24 years) and adults (25-29 and 30-35 years). We demonstrate a gradual increase in mean IVM rate in the age groups from 1 to 25 years [4.6% (1-5 years), 23.8% (6 years to menarche), and 28.4% (menarche to 17 years)], with a peak of 38.3% in the 18-24 years group, followed by a decrease in the 25-29 years group (19.3%), down to a very low IVM rate (8.9%) in the 30-35 years group. A significant difference in IVM rates was noted between the age extremes - the very young (1-5 years) and the oldest (30-35 years) groups, as compared with the 18-24-year group (p < 0.001). Importantly, number of oocytes matured, percent of patients with matured oocytes, and overall maturation rate differed significantly (p < 0.001). Our finding of extremely low success rates in those very young (under 6 years) and older (≥30 years) patients suggests that oocytes retrieved during OTC prior to chemotherapy have an optimal window of age that shows higher success rates, suggesting that oocytes may have an inherent tendency toward better maturation in those age groups.

Epigenetic changes in mammalian gametes throughout their lifetime: the four seasons metaphor.

The ability to reproduce is a major trait of living organisms. This ability is carried out by specialized reproductive cells-gametes. In mammals, gametes develop through a unique developmental pathway. Extensive changes in the epigenome of gametes occur during embryonic development. With birth, gametes continue to mature and develop until puberty. This growth process is accompanied by further epigenetic changes. When gametes mature, they reside within specialized organs-the gonads-and are exposed to both internal and external signals. The gametes' epigenome reacts to these signals, and epigenetic changes which occur can alter gene expression and the ability of the cells to go through the cell cycle. The epigenome also ages and may be one of the key players in gamete aging, which, at least for females, occurs relatively early in life. The journey gametes undertake throughout the life of the organism is thus full of epigenetic changes. In this review, we depict these changes and the mechanisms involved in them. We focus on four stages of gamete development: gametes in embryonic development, during puberty and until sexual maturity, in adulthood, and during the process of aging. In each stage, we focus on one aspect of epigenetic changes and discuss it in more detail. These four stages include many different molecular players, lots of enzymatic activity, and abrupt changes. By this, these stages resemble the four seasons of the year. Thus, we describe epigenetic changes in gametes as changes throughout four seasons of life.