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. 2011 Jul;25(7):2245–2255. doi: 10.1096/fj.10-180166

Regulation of female reproduction by p53 and its family members

Zhaohui Feng *, Cen Zhang *, Hey-Joo Kang †, Yvonne Sun *, Haijian Wang ‡, Asad Naqvi ‡, Amanda K Frank §, Zev Rosenwaks †, Maureen E Murphy §, Arnold J Levine *,‡,1, Wenwei Hu *,1
PMCID: PMC3114525  PMID: 21402718

Abstract

Tumor suppressor p53 is crucial for embryonic implantation through transcriptional up-regulation of uterine leukemia inhibitory factor (LIF). This article reports that p53 and estrogen receptor α were activated in endometrial tissues during implantation to coordinately regulate LIF production. By using human p53 knockin (Hupki) mice carrying a single nucleotide polymorphism (SNP) at codon 72 (arginine/proline), the arginine allele was demonstrated to produce higher uterine LIF levels during implantation than the proline allele. In humans, the diversity of haplotypes of the p53 gene has decreased during evolution, because the arginine allele, existing in only a subset of haplotypes, is under positive selection. This observation is consistent with previous results showing that the proline allele is enriched in patients undergoing in vitro fertilization (IVF). Studies with p63- and p73-knockout mice have demonstrated the involvement of p63 and p73 in female reproduction and their roles in egg formation and apoptosis (p63) and spindle checkpoint (p73) in female mice. Here, the role of p63 and p73 in human reproduction was investigated. Selected alleles of SNPs in p63 and p73 genes were enriched in IVF patients. These findings demonstrate that the p53 family members are involved in several steps to regulate female reproduction in mice and humans.—Feng, Z., Zhang, C., Kang, H.-J., Sun, Y., Wang, H., Naqvi, A., Frank, A. K., Rosenwaks, Z., Murphy, M. E., Levine, A. J., Hu, W. Regulation of female reproduction by p53 and its family members.

Keywords: implantation, leukemia inhibitory factor, single nucleotide polymorphism, p63, p73

The p53 tumor suppressor protein plays a crucial role in maintaining genomic stability in somatic cells and preventing tumor formation (1, 2). Recent studies have revealed a novel but very important function of p53: p53 regulates female reproduction and blastocyst implantation (3, 4). Loss of p53 in female but not male mice causes a significant decrease in fertility. A significantly lower pregnancy rate and litter size were observed when p53−/− female mice mated with p53+/+, p53+/−, or p53−/− male mice in several inbred mouse strains (3). This function of p53 is mediated by its transcriptional target gene, leukemia inhibitory factor (LIF). LIF plays a critical role in blastocyst implantation. The expression levels of LIF are increased significantly in the uterus at the onset stage of blastocyst implantation. LIF−/− female mice have a defect in reproduction due to the failure of implantation (5, 6). p53 can regulate the transcription of LIF. Loss of p53 decreases uterine LIF levels, especially at the implantation stage, which results in the impaired implantation (3). Injection of exogenous LIF at the implantation stage rescues the impaired implantation and the reproduction in p53−/− female mice (3). Implantation is the process by which the blastocyst becomes intimately connected with the maternal endometrium/decidua, which involves the concerted actions of maternal hormones.

In addition to p53, estrogen and estrogen receptor α (ERα) have been suggested to be involved in the regulation of the transient induction of uterine LIF expression at the implantation stage (6). To understand the coordinated regulation of uterine LIF expression during pregnancy, especially at the implantation stage, the spatiotemporal expression of p53 and ERα and their effect on uterine LIF levels were investigated in this study.

In humans, p53 appears to play a similar role in reproduction. As in mice, LIF has been suggested to be an important factor for implantation in humans. The levels of LIF protein are increased at the time of expected implantation in fertile women (7). Lower LIF levels have been observed in women with unexplained infertility compared with normal fertile women (8, 9). The regulation of LIF by p53 is conserved from mice to humans (3), suggesting that p53 may regulate the efficiency of human implantation and reproduction through LIF. It has been well documented that small changes in the levels or activity of p53 can alter its functional efficiency in a cell (10–12). In cells, the levels and activity of p53 are under tight regulation by a panel of regulators to ensure the proper function of p53. In humans, naturally occurring single nucleotide polymorphisms (SNPs) with functional consequences have been identified in genes at critical nodes in the p53 pathway. Among them, there is a common coding SNP in the p53 gene, which results in either an arginine or a proline at codon 72 of the p53 protein (11). The p53 arginine allele exhibits 2-fold higher transcriptional activity toward the LIF gene compared with the p53 proline allele in cultured human cells (4, 12). The p53 proline allele was found to be enriched in patients undergoing in vitro fertilization (IVF) and is a risk factor for implantation failure after IVF (4). The strong association of p53 codon 72 with infertility suggests that p53 regulates the efficiency of human fertility, and this suggestion is supported by a strong positive selection of the p53 arginine allele in the evolution of Caucasian and Asian populations that arose more recently. In this study, the effect of the p53 codon 72 SNP on uterine LIF expression was directly investigated in vivo by using human p53 knockin (Hupki) mice carrying either the arginine or the proline allele (13). The p53 codon 72 SNP was found to have a direct effect on uterine LIF levels, especially at the implantation stage, suggesting that p53 regulates the efficiency of human reproduction through its regulation of LIF.

Recent reports have revealed that p63 and p73, two members of the p53 family, are also involved in the regulation of female reproduction in mice (14–16). p63 is required to form mature eggs in the ovary (15, 17), whereas p73 plays a role in the spindle checkpoint in eggs (14, 18). To investigate whether p63 and p73 regulate human reproduction, several SNPs in human p63 and p73 genes were selected to determine their association with human fertility. Results presented here demonstrate that selected alleles of SNPs in p63 and p73 genes are enriched in IVF patients older than 35 yr, who are more likely to have decreased quality of oocytes that have higher rates of aneuploidy. Patients younger than 35 yr are more likely to have impaired implantation (a function of p53) compared with older patients. Taken together, these results strongly suggest that p53 family members are guardians of the female reproduction in mice and humans through different mechanisms.

MATERIALS AND METHODS

Animals and tissue preparation

C57BL6/J mice were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). Hupki mice containing a knock-in human p53 gene with either the p53 arginine allele or the proline allele were established as described previously (13). Six-week-old female mice were mated with fertile males of the same strain to induce pregnancy (d 1=vaginal plug). Uteri were collected from mice at different days during pregnancy.

Immunohistochemistry (IHC) and immunofluorescence staining

Uterine sections (5 μm) cut from paraffin-embedded uterine tissues were deparaffinized with xylene and rehydrated with ethanol. For IHC, tissue sections were preincubated with 10% normal rabbit or goat serum in PBS (pH 7.5) and then were incubated with primary antibodies overnight at 4°C. Tissue sections were stained with biotinylated secondary antibody (Vector Laboratories, Burlingame, CA, USA) for 1 h at room temperature. Immunoreactivity was detected using the Vectastain Elite ABC kit (Vector Laboratories) and visualized as brown staining. IHC staining was evaluated by the semiquantitative H-score method as described previously (19). The score was obtained by the formula: 3 × percentage of strongly staining cells + 2 × percentage of moderately staining cells + percentage of weakly staining cells. For immunofluorescence staining, tissue sections were boiled in Antigen Unmasking Solution (H-3300; Vector Laboratories) for 10 min for antigen retrieval. Tissue sections were preincubated with 10% goat serum in 0.25% PBS-Tween 20 and incubated with primary antibodies overnight at 4°C, followed by incubation with anti-mouse IgG (Alexa Fluor 488; Invitrogen, Carlsbad, CA, USA) and anti-rabbit IgG (Alexa Fluor 555; Invitrogen) for 1 h. Slides were mounted with mounting medium containing DAPI and analyzed immediately using a confocal laser scanning microscope (Nikon, Melville, NY, USA). Antibodies used in this study included anti-p53 antibody (Pab248) and anti-ERα (MC-20) antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-PR antibody (C-19; Abcam, Cambridge, MA, USA), and anti-LIF antibody (R&D Systems, Minneapolis, MN, USA).

Primary uterine epithelial cell culture

Mouse uterine epithelial cells from p53+/+ and p53−/− mice were isolated and cultured as described previously (20). In brief, uteri from mice were cut open lengthwise, and incubated in 0.5% trypsin, 2.5% pancreatin (bovine pancreatic, type III; Sigma-Aldrich, St. Louis, MO, USA) in PBS for 45 min at 4°C and then for 45 min at 37°C. DMEM supplemented with 10% fetal bovine serum (FBS) was added to neutralize proteases, and uteri were gently agitated by repeated pipetting to release the epithelial cells. Cells were maintained at 37°C in DMEM supplemented with 10% FBS. For estrogen and progesterone treatment, cells were cultured in phenol red-free medium supplemented with 10% charcoal-stripped FBS for 3 d before being treated with various concentrations of 17β-estradiol (Sigma-Aldrich) or progesterone (Sigma-Aldrich) for 12 h.

Quantitative real-time PCR

Total RNA was prepared from cells using an RNeasy kit (Qiagen, Valencia, CA, USA). Real-time PCR was performed in triplicate with TaqMan PCR Mix (Applied Biosystems, Carlsbad, CA, USA) in the 7000 ABI Sequence Detection System (Applied Biosystems). All primers were purchased from Applied Biosystems. The expression of target genes was normalized to that of the β-actin gene.

Western blot assay and ELISA

A standard Western blot assay was used to analyze protein expression. Anti-p53 antibody (FL393), anti-ERα antibody (MC-20), and anti-PR antibody (C-19) were purchased from Santa Cruz Biotechnology. Anti-β-actin (A5441) was purchased from Sigma-Aldrich. A mouse LIF ELISA kit (R&D Systems) was used to measure the LIF protein levels in primary cultured uterine epithelial cells and mouse tissues according to the manufacturer's instructions.

Study participants

Lymphoblastoid cell lines from the Coriell Diversity Cell Line panel

Lymphoblastoid cell lines (Caucasian 200) established from healthy Caucasian individuals (n=200) were obtained from the Coriell Diversity Cell Line panel (Coriell, Camden, NJ, USA).

Patients from an IVF clinic

Women with unexplained infertility (n=339) from the Center for Reproductive Medicine and Infertility at Weill Cornell Medical College were prospectively enrolled in this study. Of these, 173 patients were older than 35 yr and 166 patients were younger than 35 yr. Young patients with severe male factor infertility, poor ovarian reserve, ovulatory dysfunction, or müllerian anomalies were excluded from the study.

SNP genotyping and haplotype analysis

SNPs genotyped in this study include a list of 14 SNPs in the p53 gene (rs2287497, rs2287498, rs1421314, rs8064946, rs8078476, rs9897559, rs8079544, rs1642785, rs1800371, rs1042522, rs2909430, rs1625895, rs12951053, and rs9914052), 1 SNP in the p63 gene (rs2279744), and 2 SNPs in the p73 gene (rs4648551 and rs6695978). The status of these SNPs was determined by using TaqMan SNP genotyping assays. All primers were purchased from Applied Biosystems.

For haplotype analysis, Fisher's exact test was used to determine the statistical significance of fitness to Hardy-Weinberg equilibrium for each SNP in the p53 gene. Haplotypes were reconstructed from the unphased genotypes with PHASE software (21). Haplotype diversity was calculated by using DnaSP (v5) software (22).

RESULTS

Selective activation of p53 and ERα, two important regulators of LIF expression, at the implantation stage

Results from our previous study have shown that LIF is a p53-regulated gene, and p53 regulates LIF expression in the uterus (3). Uterine LIF expression levels were significantly lower in p53−/− mice than in p53+/+ mice, both before pregnancy [day 0 of pregnancy 0 (D0)] and at the implantation stage (D4; Fig. 1A). The uterus is composed of heterogeneous cell types that undergo dynamic changes to support implantation. In mice, the highest levels of LIF expression are observed in the endometrial glands at the implantation stage (Fig. 1A; ref. 6). Although it is clear that LIF expression in the uterus requires p53 functioning as a transcription factor, it remains unclear whether the regulation of LIF expression at the implantation stage requires the activation and increased levels of p53 protein or only the basal levels of p53 protein. To understand the regulation of LIF by p53 in the uterus during pregnancy, the spatiotemporal expression of uterine p53 during pregnancy was determined by IHC staining using a p53 antibody in uterine tissues collected from p53+/+ C57BL6/J mice during pregnancy. As shown in Fig. 1B, p53 protein levels in selected secretory cells were increased significantly at D4 in the endometrial tissues, including endometrial glands in which the highest levels of LIF expression were observed, demonstrating that the transcriptional regulation of LIF at the implantation stage is accompanied by increased levels of p53. These results suggest that p53 protein is selectively activated at D4 by some intracellular or extracellular signals to induce the expression levels of the p53 target LIF.

Figure 1.

Figure 1.

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IHC staining of LIF, p53, and ERα in mouse uterus during pregnancy. Uteri were collected from p53+/+ and p53−/− C57BL6/J mice at different days during pregnancy. Expression levels of LIF (A), p53 (B), and ERα (C) were examined by IHC staining. A) Increase in LIF staining in endometrial gland tissues at D4 was much more significant in p53+/+ mice than in p53−/− mice. B) In p53+/+ mice, p53 protein levels were increased significantly at D4 in the endometrial tissues, including endometrial glands. C) In p53+/+ and p53−/− mice, nuclear staining of ERα was increased significantly at endometrial gland tissues at D4. Uteri from at ≥3 mice at the same day of pregnancy were examined, and similar results were obtained. *P < 0.01 vs. p53+/+; t test.

It has been suggested that estrogen is involved in the regulation of the transient induction of LIF expression at the implantation stage (6). The increase in uterine LIF levels at the implantation stage directly parallels the increase in estrogen levels in mice. The effects of estrogen are mainly mediated through its nuclear receptor, ERα. Studies on whole uteri of primates have shown that the expression of ERα is stimulated by estrogen (23). To understand the coordinated regulation of uterine LIF at the implantation stage, the spatiotemporal expression of ERα in uterine tissues from p53+/+ and p53−/− mice was examined by IHC staining. As shown in Fig. 1C, the uteri from nonpregnant p53+/+ and p53−/− mice show clear nuclear staining in a variety of cell types, demonstrating constitutive expression of ERα in the endometrial epithelial, the stromal gland, and the myometrial compartments. At D4, there is much stronger nuclear staining of ERα in the endometrial gland tissues compared with that at D0, whereas the staining of ERα in other uterine compartments remains at a level similar to that at D0. Furthermore, very similar spatiotemporal expression patterns of ERα were observed in p53+/+ and p53−/− uterine tissues.

To directly investigate the regulation of uterine LIF expression by p53, estrogen, and ERα, primary cultured mouse endometrial epithelial cells that express ERα (Fig. 2A) were used. Mouse uterine epithelial cells isolated from 6-wk-old p53+/+ and p53−/− C57BL/6J female mice were cultured as described previously (20), and cells were treated with or without estrogen (17β-estradiol) for 12 h. The expression of LIF was determined at RNA and protein levels by using quantitative real-time PCR and ELISA, respectively. The basal RNA and protein levels of LIF in p53−/− uterine epithelial cells were one-third of those in p53+/+ uterine epithelial cells (Fig. 2B, C). Estrogen treatment significantly increased LIF expression levels in both p53+/+ and p53−/− uterine epithelial cells. However, 2- to 4-fold lower LIF levels were observed in p53−/− uterine epithelial cells. These results are similar to previous observations that uterine LIF expression levels were significantly lower in p53−/− mice than in p53+/+ mice, both before pregnancy and at the D4 implantation stage (3). These results demonstrate that the selective activation of p53 and ERα in endometrial tissues coordinately regulates LIF expression at the implantation stage.

Figure 2.

Figure 2.

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Coordinated regulation of LIF by p53, estrogen, and ERα. Primary cultured mouse uterine epithelial cells isolated from p53+/+ and p53−/− mice were treated with various concentrations of estrogen (left panels) or progesterone (right panels) for 12 h. A) Protein levels of ERα and PR were determined by Western blot assays. B) LIF RNA levels were measured by quantitative real-time PCR. All values were normalized to levels of β-actin; averages of ≥3 independent experiments are presented. C) LIF protein levels were determined by an ELISA. *P < 0.01 vs. p53+/+; #P < 0.05 vs. control; t test.

In addition to estrogen, progesterone, whose function is mediated largely by progesterone receptors (PRs), is another important hormone for pregnancy. To investigate whether progesterone is involved in the regulation of uterine LIF expression, primary cultured mouse uterine epithelial cells that express PRs (Fig. 2A) were treated with progesterone for 12 h. No apparent difference in the LIF RNA and protein levels was observed in cells with and without progesterone treatment (Fig. 2B, C), suggesting that progesterone and PRs may not be directly involved in the regulation of LIF at the implantation stage. Uterine tissues from mice at D0 and at D4 were also stained by immunofluorescence with antibodies against PRs and p53 to examine the spatiotemporal expression of PRs and p53. Some cells with high levels of p53 were also positive for PR staining, but some endometrial glandular cells with high expression of p53 were negative for PR staining (Fig. 3). This observation helps to explain some conflicting results published by Hirota et al. (24). Results from the study of Hu et al. (3) showed that p53−/− mice containing no wild-type p53 protein in any tissues failed to make normal levels of LIF and that there was a 4- to 6-fold reduction in LIF levels in the uterus (3). Hirota et al. (24) used a lox p53 gene in mice and used a PR promoter to activate the Cre gene that inactivates p53 in those selected tissues. They detected only a 2-fold reduction in LIF levels and failed to observe implantation failure in their mice but instead observed uterine senescence later in pregnancy that could have been mediated by p53 (24). The presence of p53-positive, ERα-positive but PR-negative cells in the uterus explains why residual p53 activity (and LIF activity) was present for implantation in their experiments. This unregulated p53 activity in the PR-Cre-lox mice could be responsible for a well-known p53 function, senescence, observed later in pregnancy with their mice (24).

Figure 3.

Figure 3.

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Expression of uterine p53 and PR at the implantation stage. Uteri were collected at D0 (top panel) and D4 (bottom panel) from p53+/+ C57B/6J mice. Expression levels of p53 and PR were examined by immunofluorescence staining.

p53 codon 72 SNP has differential transcriptional induction of uterine LIF in mice

The regulation of LIF expression by p53 is conserved from mice to humans, and the function of p53 in female reproduction appears to be conserved in humans as well (4). Our previous study has shown that the p53 proline allele is enriched in IVF patients and is a risk factor for implantation failure in these patients (4). IVF patients homozygous for the proline allele have a significantly lower implantation rate and pregnancy rate after an IVF procedure than patients carrying at least 1 arginine allele. However, it is unclear whether the association of p53 codon 72 SNP with implantation and human fertility is due to the differential regulation of uterine LIF by p53 codon 72 SNP. To directly investigate the effect of p53 codon 72 SNP on uterine LIF expression levels in vivo, especially at the implantation stage, which may contribute to different reproduction efficiency, Hupki mice carrying either the p53 arginine allele or the proline allele were used. The Hupki mouse contains exons 4–9 of the human p53 gene in place of the corresponding mouse exons and retains many wild-type p53 functions in vivo (25, 26). LIF expression at RNA and protein levels was determined in uterine tissues from Hupki mice at D0 or D4 by using quantitative real-time PCR and ELISA, respectively. As shown in Fig. 4A, Hupki mice carrying either the p53 arginine allele or proline allele have similar p53 protein levels. Uterine LIF expression levels are much higher at D4 than at D0. RNA levels of other p53 target genes, including p21 and MDM2, were not significantly increased at D4 (Fig. 4B). Interestingly, LIF expression levels are lower in Hupki mice carrying the p53 proline allele compared with mice carrying the arginine allele, both at D0 and D4 (Fig. 4B, C). These results demonstrate that the p53 proline allele produces less uterine LIF in mice, especially at the implantation stage, which may contribute to the increased risk of implantation failure and infertility in humans observed in IVF patients carrying the p53 proline allele (4).

Figure 4.

Figure 4.

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Regulation of uterine LIF expression levels in Hupki mice carrying different SNPs at codon 72. Uteri were collected at D0 or D4 from Hupki mice carrying either the p53 proline allele (P72) or the arginine allele (R72). A) p53 protein levels were determined by Western blot assays. B) RNA levels of LIF and other p53 target genes, including p21 and MDM2, were measured by quantitative real-time PCR. All values were normalized to levels of β-actin; averages of results from ≥3 mice/group are presented. C) LIF protein levels were determined by ELISA. *P < 0.05 vs. P72; t test.

Diversity of haplotype structures of the p53 gene in different ethnic backgrounds

Our previous study demonstrated that several SNPs that regulate the levels and activities of the p53 pathway, including p53 codon 72 SNP, are associated with human fertility, which strongly suggests that p53 regulates the efficiency of human reproduction (4). The efficiency of reproduction exerts strong evolutionary selection pressure. Therefore, it is possible that certain haplotypes containing SNPs that have an effect on human reproduction, such as codon 72 SNP, are under positive evolutionary selection pressures. To test this hypothesis, the haplotype structure of the p53 gene was determined in Caucasian and African populations. Fourteen SNPs in the p53 gene were genotyped in 96 healthy Caucasian individuals and 96 healthy African individuals from the Coriell Diversity Cell Line panel. Haplotypes were reconstructed from the unphased genotypes using PHASE software, and a haplotype diversity index was calculated based on haplotype frequencies using DnaSP (v5) software. In Africans, the p53 gene has 45 haplotypes and a haplotype diversity index of 0.943, whereas in Caucasians, a population who arose more recently, the p53 gene has 15 haplotypes and a haplotype diversity index of 0.542 (Table 1). One reason for the haplotype diversity index being so low in Caucasians is that an allele that is under strong positive selection in the Caucasian population arose (the arginine allele) in the recent past (3–5×104 yr), and this limits the number of haplotypes that persist in the population before recombination, which generates additional haplotypes. A similar pattern of limited haplotype diversity was observed in the p53 gene in Caucasian and Asian populations collected in the HapMap 3 database (Table 1). The haplotype diversity index of the p53 gene in Africans (YRI, Yoruba in Ibadan, Nigeria) is 0.843. Caucasians (CEU, Utah residents with Northern and Western European ancestry from the CEPH collection) have a diversity of 0.489, and Asians have a diversity index of 0.748. There is a strong selection of certain haplotypes in Caucasians, and to a lesser extent in Asians, with little loss of diversity in Africans (Table 1). These results support the hypothesis that haplotypes containing SNPs regulating reproductive efficiency are under recent evolutionary selection pressures in Caucasian and Asian populations.

Table 1.

Haplotype diversity of the p53 gene in human populations

Source Population Sample size (n) SNP number Haplotype number Haplotype diversity
Coriell African 96 14 45 0.943
Caucasian 96 12 15 0.542
HapMap phase 3 African 113 7 15 0.843
Caucasian 113 7 10 0.489
Chinese 84 9 11 0.748
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African population (African American panel of 100) and Caucasian population (Caucasian panel of 100) collected by Coriell Cell Repositories, and three major populations in HapMap phase 3: Africans (YRI, Yoruba in Ibadan, Nigeria), Caucasians (CEU, Utah residents with Northern and Western European ancestry from the CEPH collection), and Asians (CHB, Han Chinese in Beijing, China) were used. Haplotype diversity, the expected heterozygosity based on haplotype frequencies, was estimated using DnaSP (v5) software (22).

SNPs in the p63 and p73 genes are associated with infertility

Recent studies in mice have revealed that other p53 family members, including p63 and p73, are all involved in female reproduction (16). p63 and p73 are alternatively spliced and have multiple isoforms. The full-length isoforms (TAp63 and TAp73), which contain a transactivation domain, share similarity with p53 in overlapping target genes and biological functions (27). Female mice deficient for TAp63 or TAp73 are infertile, whereas males are not (14, 15). p63, in particular TAp63, is highly expressed in female germ cells during meiotic arrest and plays an important role in DNA damage-induced apoptosis in female germ cells (15, 17). p73 also has a distinct and direct role in female reproduction. The size of the primordial follicle pool is crucial for reproductive abilities in humans. TAp73-deficient mice have significantly smaller numbers of primordial and primary follicles (14). The ovulation rate is significantly lower in TAp73-deficient mice. More important, TAp73 regulates spindle assembly checkpoint functions during meiosis and mitosis (18, 28). Oocytes from TAp73-deficient mice have a striking increase in spindle abnormalities, aneuploidy, and poor developmental competence. These findings in mice clearly demonstrate the important roles of p63 and p73 in female reproduction through maintaining the fidelity of female germ line cells and ovary function.

Based on these observations, we investigated whether p63 and p73 are involved in human fertility by exploring whether polymorphisms in human p63 and p73 genes could be involved in reproductive deficits. A list of SNPs in the p63 and p73 genes was selected to determine their association with human fertility. These SNPs include a SNP in the p63 gene (rs17506395) and two SNPs in the p73 gene (rs4648551 and rs6695978), which appear to be under evolutionary selection pressures using the criteria of Atwal et al. (29, 30) and information theory.

The frequency of these SNPs was determined in 339 Caucasian IVF patients with unexplained infertility, recruited at Weill Cornell Medical College. Of these, 166 patients who were younger than 35 yr underwent an IVF procedure using their own oocytes, and 173 patients who were older than 35 yr underwent donor IVF procedures using oocytes from donors of younger age to avoid the contribution of aneuploidy to infertility, which is associated with increasing maternal age. As controls, 200 healthy Caucasian individuals from the Coriell Diversity Cell Line panel were used. There is a clear enrichment of selected alleles of these SNPs in IVF patients (Table 2). Interestingly, the association of SNPs in the p73 gene (spindle checkpoint) with infertility was observed only in older patients, who are more likely to have decreased quality of oocytes with chromosomal aneuploidy, but not in young patients. The enhanced frequency of an allele in the tested p63 SNP (egg production and apoptosis) was observed in young (<35 yr) and older patients. These results demonstrate the association of SNPs in the p63 and p73 genes with human infertility, which strongly suggests the involvement of these p53 family members in human fertility similar to the observations made in mice.

Table 2.

Significant enrichment of selected alleles of p63 and p73 genes in IVF patients

Gene and SNP Genotype of SNP Control [n (%)] IVF patients
<35 yr
 ≥35 yr
 Total
n (%) Pa n (%) P n (%) P
p63
    rs17506395 TT 101 (52.1) 101 (60.8) 0.05b 112 (64.7) 0.04b 213 (62.8) 0.02b
TG 73 (37.6) 58 (34.9) 50 (28.9) 108 (31.9)
GG 20 (10.3) 7 (4.3) 11 (6.4) 18 (5.3)
T 275 (70.9) 260 (78.3) 0.02b 274 (79.2) 0.01b 534 (78.8) 0.004b
G 113 (29.1) 72 (21.7) 72 (20.8) 144 (21.2)
p73
    rs4648551 GG 67 (34.4) 62 (37.3) 0.82 75 (43.4) 0.02b 137 (40.4) 0.21
GA 88 (45.1) 70 (42.2) 80 (46.2) 150 (44.2)
AA 40 (20.5) 34 (20.5) 18 (10.4) 52 (15.3)
G 222 (56.9) 194 (58.4) 0.70 230 (66.5) 0.004b 424 (62.6) 0.04b
A 168 (43.1) 138 (41.6) 116 (33.5) 254 (37.4)
    rs6695978 GG 177 (92.2) 136 (89.5) 0.39 135 (84.9) 0.09 271 (87.1) 0.17
GA 14 (7.3) 16 (10.5) 23 (14.5) 39 (12.6)
AA 1 (0.5) 0 (0) 1 (0.6) 1 (0.3)
G 368 (95.8) 288 (94.7) 0.31 293 (92.1) 0.03b 581 (94.2) 0.07
A 16 (4.2) 16 (5.3) 25 (7.9) 41 (6.6)
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a

χ2 test.

b

Significant difference observed between IVF patients and control subjects.

DISCUSSION

The p53 family of genes (p53, p63, and p73) is conserved over evolutionary time scales (31). The p53, p63, and p73 proteins form a family of transcription factors that are involved in many important processes, including tumor prevention and development (32). The primordial functions of the p53 ancestral genes are to ensure germ line genomic integrity and the fidelity of the developmental process to produce normal offspring that will survive or reproduce. In vertebrates, the p53 family of genes retains those functions in germ line genomic integrity but has added important functions in regulation of reproduction and development. p53 regulates implantation of the blastocyst through transcriptional regulation of LIF. p63, in particular TAp63, is a main regulator of the process that controls female germ line fidelity by an elimination (apoptotic) mode of protection. TAp63 is phosphorylated on DNA damage, which in turn monitors the repair of DNA damage and induces p53-independent apoptosis of oocytes to protect the quality of the female germ lines during meiotic arrest (15, 33). p73 plays a crucial role in maintaining the size of the primordial follicle pool and ovulation rate, as well as acting at the spindle checkpoint, reducing aneuploidy in the offspring. Loss of p53, p63, or p73 genes in female mice leads to a significant decrease in fertility.

Results from mouse studies clearly demonstrate that p53 plays an important role in blastocyst implantation, which is mediated by LIF. LIF is a multifunctional cytokine that plays a crucial role in implantation, with its levels transiently induced at the implantation stage (34). p53 transcriptionally regulates the expression levels of LIF. It was unclear whether the regulation of uterine LIF expression at the implantation stage requires the activation of p53 and an increase in its protein levels. In our previous effort to study uterine p53 protein levels at the implantation stage, we used Western blot assays and did not observe a significant change in p53 protein levels. In this report, the spatiotemporal expression of p53 protein was determined during pregnancy in mice by IHC staining, and selective activation of p53 protein in the endometrial region was observed at the implantation stage. This result strongly suggests that the transient increase in LIF expression levels at implantation requires the increased levels and activation of the p53 protein, although the signals that activate p53 in endometrial tissues at the implantation stage are currently unclear. Normal gestation implantation occurs under the hypoxic condition (35). Hypoxic signals are known to activate p53 transcription (36), which might be the stimulus in the uterus.

The preparation of the uterus for implantation requires coordinate effects of estrogen and progesterone through ERs and PRs. It has been reported that LIF can be regulated by estrogen (3, 6). Estrogen levels are increased significantly at the implantation stage in mice. Estrogen binds to ERα, a ligand-inducible transcription factor, to modulate the expression of their target genes. Here, using IHC staining, we observed a significant increase in nuclear ERα levels in endometrial glands at the implantation stage. The coordinated regulation of LIF by estrogen, ERα, and p53 was further demonstrated in primary cultured mouse uterine epithelial cells. Taken together, these results suggest that the increased expression of LIF at the implantation stage requires the activation of p53, increased estrogen levels, and activated ERα.

Progesterone is another important pregnancy hormone involved in implantation. Previous studies on the spatiotemporal expression of PRs using IHC staining have shown that nuclear staining of PRs at the implantation stage was mainly in the luminal epithelium and the subepithelial stroma and was below the level of detection in endometrial glands (37), suggesting that progesterone and PRs may not be directly involved in the regulation of LIF at the implantation stage. Consistent with previous reports, immunofluorescence staining of PRs and p53 protein in the uterus showed that there is a subset of uterine cells with undetectable levels of PRs, including cells with p53 activation (Fig. 3). Hirota et al. (24) used PR-Cre transgenic mice crossed with floxed p53 mice to generate a conditional deletion of uterine p53, but, as demonstrated here, this does not eliminate all p53 protein expressed in glandular cells in the uterus. By using this system to study the function of p53 in reproduction, only a 2-fold decrease of LIF expression levels was observed in the uterus, and these mice still had normal implantation (24), which was probably due to the residual uterine p53 protein and LIF protein in these mice.

In humans, the p53 family of genes appears to retain its roles in reproduction. Codon 72 SNP in the p53 gene, a functional SNP that affects the transcriptional activity of p53 toward some of its target genes, has a direct effect on uterine LIF expression levels at the implantation stage. The p53 proline allele, which produces lower uterine LIF levels, is enriched in IVF patients and is a risk factor for implantation failure in Caucasians (4). Of interest, the frequency of codon 72 SNP differs greatly in populations with different ethnic backgrounds. The p53 proline allele, which appears to be the ancestral allele, has a >60% frequency in Africans but becomes the minor allele in Caucasians and Asians (11). The allele structures in the p53 gene show different levels of haplotype diversity in populations with different ethnic backgrounds, suggesting that certain alleles are under evolutionary selection pressures, depending on the ethnic background. What is the cause of this selection in Caucasians and Asians? Reproduction is much more likely to be the reason for the observed evolutionary selection pressure. It is unlikely that cancer incidence plays a significant role in evolutionary selection because it arises mostly late in life, after the reproductive years. Indeed, our previous study has shown that selected alleles in several genes in the p53 pathway, which are under evolutionary selection, are strongly associated with human fertility (4). In this study, selected alleles of SNPs in p63 and p73, which also appear to be under evolutionary selection, are enriched in IVF patients, suggesting that the genes of p53 family members regulate the efficiency of fertility in humans. Of interest, the at-risk alleles in the p53 pathway for human fertility mainly act in young patients (<35 yr), and the at-risk alleles in the p73 gene for human fertility act in older patients. The at-risk allele in the p63 gene for human fertility is found to be enriched in both older and young patients. It is well known that maternal age has a significant negative effect on fertility, and a major underlying mechanism for infertility in older patients is aneuploidy of oocytes and poor quality of embryos. It is likely that in patients older than 35 yr, factors such as aneuploidy may predominate, whereas in young patients, factors such as impaired implantation may play a more significant role in infertility. The enrichment of SNPs in the p53 family of genes in patients with different causes of infertility suggests that the p53 family of genes regulates the efficiency of human fertility through different mechanisms. p53 regulates blastocyst implantation, and p63 and p73 regulate ovary functions and female germ cell integrity in humans. The association of SNPs in p63 with infertility in both young and older patients suggests that p63 may have other functions in reproduction in addition to its role in maintaining female germ cell integrity.

Acknowledgments

This study was supported by U.S. National Institutes of Health grant 1P30-CA147892-01 (to W.H. and A.J.L.).

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