TAp73 is downregulated in oocytes from women of advanced reproductive age

Abstract

Studies on oocyte transcriptome are important to understand the biological pathways involved in oogenesis, totipotence and early embryonic development. Moreover, genes regulating physiological pathways in gametes could represent potential candidates for reproductive disorders. In addition to oocyte specific transcription factors, also the members of the p53 family could be etiologically involved due to their biological functions. In fact, their role in the control of cell cycle, apoptosis and germ-line genome stability is well known. Female reproductive aging is one of the causes of fertility reduction and it is often associated with egg aneuploidy increase. In order to verify the potential involvement of p73 in reproductive aging, we determined its expression in single mature MII oocytes from two groups of women, younger than 35 or older than 38 y, respectively. We found that TAp73 isoforms are downregulated in oocytes from women older than 38 y. We confirmed these data in pools of mouse oocytes. TAp73 downregulation in oocytes from women of advanced reproductive age could explain both the reduction of fertility and the increase of newborns with chromosomal abnormalities.

Introduction

Mature oocytes are highly differentiated cells, nevertheless they are able to give rise to a new individual after fertilization; also, their cytoplasm is capable of reprogramming the nucleus of somatic cells.¹ During their maturation, oocytes synthesize and store maternal RNAs that are important for their own differentiation or will be used during early embryonic development. The identification of genes that are essential for oogenesis, fertilization and early embryonic development is important to comprehensively understand reproductive and developmental biology. Several studies have led to the identification of oocytespecific transcription factors, which are required for germ cell development and embryonic transcription as FIGLA (Factor In the GermLine), NOBOX (Newborn Ovary Homeobox Gene), GDF9 (Growth Differentiation Factor 9) and its paralog GDF9B, MATER (Maternal Antigen That Embryos Require), ZAR1 (Zygote Arrest 1).^2–5 In parallel with these oocyte STFs (Specific Transcription Factors), we found that also GTFs (General Transcription Factors) as TAF4B (TBPassociated factor 4B) and TBPL2 (TATA box binding protein like 2) could play an important role during oogenesis and in the activation of embryonic transcription.^6–7 The genes encoding both GTFs and STFs perform a fundamental role in oocyte differentiation:^8,9 accordingly, they represent valuable candidates for reproductive disorders.¹⁰ Because of their well known involvement in the control of cell cycle, apoptosis, genome stability including ploidy, also the members of the p53 family (p53, p63, p73) could be etiologically involved in these disorders.^11,12 Female reproductive aging is the process dictated by a gradual decrease in both the quantity and the quality of the oocytes. The pool of ovarian follicles is progressively reduced during female reproductive lifespan, which ends with menopause at a mean age of 50–51 y. With the decay in follicle numbers, oocyte quality also diminishes: in fact, fertilization rates decrease while the frequency of miscarriages increases.¹³ Also, during in vitro fertilization cycles older women tend to produce less oocytes after hormonal stimulation and derived embryos have a lower implantation potential.¹⁴ The existence of ovarian stem cells, able to sustain oocyte and follicle production in the adult ovary, has been convincingly demonstrated in the literature.^15,16 Even if de novo oogenesis in humans has never been demonstrated so far, due to obvious problems with in vivo studies, different molecular data seem to confirm the possibility of oocyte generation in the adult mouse.^15,16 According to this theory, a small number of ovarian stem cells remains within the ovary throughout the lifespan of the adult female; during aging, these premeiotic stem cells loose their competence to produce oocytes because they don't find a suitable microenvironmental niche.^17–19 Therefore, the decrease of oocyte quantity during aging could be determined not only by the diminished ovarian reserve, but also by germ stem cells inability to produce new oocytes. For what concerns the decrease of oocyte competence to produce a vital embryos during aging, chromosomal abnormalities are a prominent example of defects causing implantation failure. Several hypothetic mechanisms have been proposed to explain the increase of aneuploidy with aging, as defects in chromosome recombination or of chromosome cohesion at meiosis. An additional cause may be the long time interval between protracted meiotic arrest stage (known as dictyate) and MI restart, which could make oocytes unable to detect and correct recombination errors arisen at prophase during fetal life.²⁰

p53 Family as Guardian of Female Reproduction

The role of the p53 family as guardian of maternal reproduction has been recently investigated. This family consists of three members (p53, p63 and p73), conserved from invertebrates to mammals.²¹ The roles of the p53 gene and its encoded protein as tumor suppressor and guardian of the genome have been firmly established.²² Maintenance of germ-line genomic integrity is a major identified function of these proteins in Caenorhabditis elegans and Drosophila melanogaster.²¹ In vertebrates, including Homo sapiens, these proteins have been suggested to also regulate maternal reproduction.¹² Recently, p53 role in regulating embryo implantation by transcriptionally controlling the expression of LIF (Leukemia Inhibitory Factor) was demonstrated.²³ Moreover, loss of the p53, p63 and p73 genes in female mice leads to a significant decrease of fertility.¹² Finally, single nucleotide polymorphisms (SNPs) in both human p53 and p73 genes have been associated to women reproductive disorders.²⁴ Based on these data, we sought to determine p73 expression during reproductive aging.

TAp73 in Human Reproductive Aging

In order to verify the potential involvement of p73 in women reproductive aging, we determined its expression in single mature MII oocytes from two groups of women, younger than 35 or older than 38 y, respectively. We searched both quantitative expression differences of TAp73 and also ΔNp73 transcript variants. In fact, the p73 gene encodes two major groups of protein isoforms, endowed with opposite biological functions, which are synthesized through the use of alternative promoters.²¹ Proapoptotic TAp73 activates a set of p53-responsive genes and induces cell cycle arrest or apoptosis in response to DNA damage.²¹ ΔNp73 isoforms lack the N-terminal transactivation domain (TA) and perform a dominant negative role on proapoptotic isoforms.²¹ They are frequently upregulated in various human cancers, including myeloid leukemia, neuroblastoma, ovarian cancer.²⁵ Moreover, the truncated isoforms perform an important role in brain development.²⁵ In this study, we analyzed by RT-PCR Real Time 16 MII oocytes, 10 from women younger than 35 y of age and 6 from women older than 38 y of age, who had undergone intracytoplasmic sperm injection (ICSI) in an in vitro fertilization center. According to the Italian law, no more than three embryos can be produced and transferred, so we used supernumerary oocytes after the patients' informed consent and the agreement by the Hospital Ethical Committee. To avoid factors that could alter the oocyte transcriptome, we analyzed only mature MII oocytes with good morphology, which had been taken from women whose primary infertility was due to a male factor; accordingly, we excluded pathologies that could influence oocyte quality, as endometriosis, ovarian insufficiency, polycystic ovaries. We applied our previously published protocol for human oocyte RNA isolation and RT-PCR reactions.²⁶ We performed five different PCR reactions per single oocyte and repeated the same analysis in every sample. Primer sequences and RT-PCR condition are reported in Table S1. We found out that TAp73 isoforms are significantly downregulated in oocytes from women of advanced reproductive age; ΔN isoforms are expressed in human oocytes, but their expression is age independent (Fig. 1). To confirm these data, we performed a similar analysis in pools of mouse oocytes (Table S1). FVB mice (from the Animal Facility of IST) were split in two age groups: younger than 5–8 weeks (young) and older than 6–7 mo (old). Housing and treatment of animals were in accordance with the Italian and European Community guidelines and approved by the internal Ethic Committee. We found a significant TAp73 downregulation in the older cohort (Fig. 1). To verify if it were dependent on epigenetic modifications, we performed promoter methylation analysis (Table S1). Methylated CpG sites within TAp73 promoter were less than 5% in both groups: accordingly, promoter methylation doesn't explain the low levels of TAp73 expression in old mice (Fig. 1). Histone modifications during oocyte development could influence chromatin remodelling and determine specific gene expression patterns: in fact, histone acetylation, methylation, phosphorylation have been detected during mammalian oocyte maturation.²⁷ It was also suggested that the high incidence of aneuploidy in embryos from older Mus musculus females may be due to inhibition of meiotic histone deacetylation.²⁸ Finally, recent data on RNA silencing during oogenesis and early oocyte development have revealed the critical role of microRNAs in post-trascriptional gene regulation in this system.^29,30

Figure 1.

(A) The histogram shows the fold change (RQ) mean as the ln 2^−ΔΔCt in single human oocytes. We calculated the relative levels of mRNA expression in each sample according to the 2^−ΔΔCt method, using HPRT for normalization and young oocytes as calibrators. Y error bars represent variability among different samples, showing the maximum and minimum FC values. To statistically evaluate the results, young and old oocytes ΔCt were analyzed by unpaired (two sample) Student's t-test. (B) The histogram shows the fold change (RQ) mean as the ln 2^−ΔΔCt in pools of mouse oocytes. The experiment was performed in triplicate and the data are reported as mean ± SD (C). Methylation analysis was performed by bisulfite conversion and pyrosequencing to detect the methylation status of 6 CpGs in the CpG island, located at the promoter region of mouse TA p73 gene. The image shows that methylated CpG sites were less than 5% in both young (1) and old (2) mouse oocytes. Each sample was analyzed in duplicate, including an internal control, to measure the extent of bisulfite modification.

Conclusion

To the best of our knowledge and possibly as a consequence of the limited availability of samples, our data are the first on TAp73 downregulation in human old oocytes: they integrate other existing literature results and suggest an important role for this member of the p53 family as guardian of maternal reproduction in mammals. In fact, TAp73^-/- mice show defects in spindle organization during mitosis and meiosis, develop tumors and produce oocytes that even if fertilized generate non-vital embryos.³¹ A recent paper reports TAp73 interactions with the kinetochore proteins Bub1, Bub3, BubR1 components of the spindle assembly checkpoint (SAC) complex.³² If the chromosomes are not properly attached to the spindle, this complex stops the anaphase-promoting complex (APC) by negatively regulating CDC20. Absence of TAp73 removes this cell cycle brake, so causing genomic instability. Consequently, TAp73 downregulation in oocytes from women of advanced reproductive age could lead to aneuploidy in the developing embryos, explaining both the reduction of fertility and the increase of newborns with chromosomal abnormalities. At any rate and in spite of several literature data, the events determining specific mRNA phenotypes in mature oocytes are not yet clarified. For example, it is known that oxidative stress plays a significant role determining the molecular phenotype of aged ovaries.³³ Major efforts will be required to analyze the complex interactions among the different molecular players and to identify functional links among the different factors: this should allow to understand how the ovarian microenvironment during aging can influence the oocyte transcriptome and proteome and characterize the molecular bases of female aging.