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Transcript analysis identifies differential uterine gene expression profile beyond the normal implantation window in mice
Accepted Manuscript Transcript analysis identifies differential uterine gene expression profile beyond the normal implantation window in mice Qiliang Xin, Meiling Li, Haibin Wang, Sheng Cui, Guoliang Xia, Shuangbo Kong PII:
S0093-691X(17)30390-4
DOI:
10.1016/j.theriogenology.2017.08.005
Reference:
THE 14225
To appear in:
Theriogenology
Received Date: 11 November 2016 Revised Date:
3 August 2017
Accepted Date: 3 August 2017
Please cite this article as: Xin Q, Li M, Wang H, Cui S, Xia G, Kong S, Transcript analysis identifies differential uterine gene expression profile beyond the normal implantation window in mice, Theriogenology (2017), doi: 10.1016/j.theriogenology.2017.08.005. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Revised
ACCEPTED MANUSCRIPT Transcript analysis identifies differential uterine gene expression profile
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beyond the normal implantation window in mice
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Qiliang Xina,1, Meiling Lia,1, Haibin Wangb, Sheng Cuia, Guoliang Xiaa,*,
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Shuangbo Kongb,*
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a
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China Agricultural University, Beijing 100193, PR China;
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b
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University, Medical College Xiamen University, Xiamen, Fujian 361102, PR
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China.
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State Key Laboratory of Agro-biotechnology, College of Biological Sciences,
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Reproductive Medical Center, The First Affiliated Hospital of Xiamen
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Authors contributed equally to this work.
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Correspondence: Shuangbo Kong (Email: [email protected]) or
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*
Guoliang Xia (Email: [email protected])
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Running title: Uterine gene expression beyond implantation window
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Competing Interest: The authors declare no conflict of interest. 1
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Abstract Uterine receptivity is defined as a state when the uterine milieu is favorable
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for blastocyst implantation and it can only last for a limited time period. In this
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study, by utilizing the embryo transfer model, it was observed that a portion of
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blastocysts could initiate implantation even when transferred beyond the timing
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of normal uterine receptivity, while their mid-gestational embryo development
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exhibited severe retardation, suggesting that the uterine status beyond the
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normal implantation window is unconducive for normal implantation. We
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further performed microarray analysis to explore the molecular basis that
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distinguishes the normal and defective uterine receptivity. A total of 229 genes
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was found to be differentially expressed, and a large amount of them were
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epithelium-expressing genes and responsive to progesterone signaling.
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Keywords:
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progesterone-responsive genes
receptivity;
microarray;
epithelium;
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uterine
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Abbreviations: LE, Luminal epithelium; PSP, Pseudo-pregnancy; P4,
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Progesterone; E2, Estrogen; IS, Implantation site; Cyp26a1, Cytochrome P450
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26A1; PR, Progesterone receptor.
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1. Introduction Embryo implantation is one of the crucial steps in mammalian embryo
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development. In placental mammals, uterine sensitivity to implantation is
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classified into pre-receptive, receptive and non-receptive (refractory) phases
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[1-3]. In normal pregnancy, when implantation-competent blastocysts are
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ready to initiate implantation, the uterus differentiates into an altered state
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called uterine receptivity, which sustained for a limited time period. In mice, the
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normal uterine receptive phase is on day 4 of pregnancy or pseudo-pregnancy
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(day 1 = day of vaginal plug). At this stage, the uterine environment is
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conducive to blastocyst growth, attachment and subsequent events of
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implantation. In contrast, the uterine environment is unfavorable to blastocyst
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survival in the refractory phase after day 5 of pregnancy [1, 4]. Embryo
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implantation is a dynamic developmental event that involves a series of
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physical and physiological interactions between the blastocyst trophectoderm
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and various endometrial cell-types, including both the uterine epithelial and
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stromal compartment. It has been observed that the ultrastructure of luminal
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epithelium (LE), such as LE cell surface components, lateral adherent
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junctions and gap junction channels, need remolding during the establishment
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of uterine receptivity. The morphological transformations are likely associated
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with the differential gene expression in the LE, which would affect the
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ACCEPTED MANUSCRIPT trophectoderm-uterine epithelium crosstalk that fosters the normal blastocyst
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implantation. In response to the implanting embryo, the surrounding uterine
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stroma undergoes extensive cellular proliferation and transformation, a
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process known as decidualization, to accommodate embryonic growth and
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invasion. Decidualization can also occur in response to artificial stimulus by oil
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injection or endometrium scratch in the receptive uterus to form deciduoma.
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In mice, progesterone (P4), coordinating with estrogen (E2), is obligatory
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for uterine preparation for a receptive state [1, 5-7]. In mice, there are two main
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P4 receptor isoforms as PRA and PRB, arising from alternative promoter
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usage in the same gene Pgr [8]. The P4 mainly functions through its nuclear
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receptor PRA to coordinate the molecular networks for the establishment of
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uterine receptivity in mice. Numerous defects in female mice lacking the Pgr
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gene that encodes PR have been demonstrated, including failure in ovulation,
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mammary gland development, and embryo implantation along with uterine
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hyperplasia and inflammation, reflecting the critical role of P4/PR pathway in
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female reproduction [9]. Furthermore, the molecular and genetic evidence has
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also indicated that locally produced signaling molecules in the uterus, including
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cytokines, homeobox transcription factors, coordinate with these hormone
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receptors and serve as autocrine, paracrine and juxtacrine factors to specify
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the uterine receptivity [8].
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portion of blastocysts can initiate implantation even when transferred on day 5
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beyond the normal timing of uterine receptivity, while the mid-gestational
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development of these embryos exhibited severe retardation, suggesting that
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the uterine status on day 5 of pseudopregnancy (PSP) is unconducive for
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normal implantation. So we inferred that critical physiological changes occur in
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the uteri between days 4 and 5 of PSP. In order to explore the underlying
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molecular differences between these two different uterine states, we applied
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the microarray approach to analyze the uterine gene expression profile on day
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4 versus day 5 of PSP. Using this approach, here we show that, 229 out of
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about 24, 000 genes examined were significantly differentially expressed
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between days 4 and 5 PSP uteri. Further analyses revealed that a large
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proportion of (46.7%) differentially expressed genes were uterine epithelium-
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expressing genes and responsive to P4 signaling, which may compromise the
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epithelial cell differentiation program and thus the normal implantation.
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2. Materials and Methods:
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2.1 Mice
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Adult 8 weeks CD-1 male and female mice used in this study were
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purchased from the Vital River Laboratory Animal Technology Co. Ltd. All
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animal experiments were performed in accordance with the guidelines and
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regulations of the Institutional Animal Care and Use Committee of China
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Agricultural University. Adult female mice were mated with intact or
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vasectomized males of the same strain to induce pregnancy or PSP,
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respectively. The morning of seeing a vaginal plug was defined as day 1 of
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pregnancy or PSP.
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2.2 Embryo Transfer and examination of pregnancy outcome
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Day 4 blastocysts from the normal pregnant female donors were
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transferred to the uteri of recipients in the morning (10:00 h) on days 4–6 of
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PSP. Recipient mice were sacrificed to examine the implantation status at
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10:00 h on indicated days. The number of implantation site (IS) was recorded
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by tail vein injection (0.1 ml/mouse) of Chicago Blue dye (Sigma-Aldrich,
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C8679) solution (1% in saline) at 48 hours after embryo transfer. Mice were
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the uterine horns since the vascular permeability is increased at the
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implantation sites [10, 11]. In the mice without implantation sites, the uterine
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horns were flushed with saline to recover the unimplanted blastocysts. Mice
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without implantation sites but no recovery of blastocysts were excluded from
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the experiments. Recipients were examined for subsequent developmental
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events on day 12 or observed for delivery of pups at term. For each time point,
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at least 5 mice were included for the analysis.
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2.3 Experimentally induced decidualization in pseudo-pregnant mice To induce artificial decidualization, 25 µl of sesame oil (Sigma-S3547) was
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infused intraluminally in one uterine horn on days 4–6 of PSP. The
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contra-lateral horn served as control. At least 4 mice were examined in each
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time point for this experiment. Mice were sacrificed 96 hours later. Uterine
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weights of the infused and non-infused (control) horns were recorded. Fold
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increases in uterine weights were used as an index of decidualization [12].
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2.4 RNA extraction, amplification, labeling and hybridization For days 4 and 5 PSP mouse uteri (n=4 mice per group), the samples 7
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reagent (Invitrogen-15596018, Carlsbad, CA) according to manufacturer’s
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instructions. The RNA was cleaned up with RNeasy Kit (Cat #74104, Qiagen,
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Hilden, Germany) and the quantities and qualities were determined by
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spectrophotometer and 1% formaldehyde denaturing gel electrophoresis.
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Affymetrix GeneChip mouse Genome Array, which contains more than 45,000
sets
representing
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characterized mouse genes, was used in microarray analysis. Hybridization,
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data capture, and analysis were performed by Capital Bio Corporation (Beijing,
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China), a service provider authorized by Affymetrix Inc. (Santa Clara, CA).
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of
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2.5 Microarray Data Analysis
All the data were viewed pre-normalized by tree clustering to remove
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outlying microarray data, leaving three arrays (three replicates) per time point.
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The hybridization data were analyzed using Affymetrix GeneChip
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Command Console Software (AGCC), which uses statistical criteria to
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generate a ‘present’ or ‘absent’ call for genes represented by each probe set
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on the array. Afterwards, genes with ‘absent’ scores were filtered out and the
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remaining genes were analyzed. Microarray data were normalized using the
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package (http://www.bioconductor.org) was used to calculate expression
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values, which refer to the quantile normalization of Robust Multiarray Average
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method (each performed at the individual probe level). Significance Analysis of
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Microarrays (SAM) was used to identify genes that are differentially expressed.
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[13].
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2.6 Validation of microarray data by Real-time PCR
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Differentially expressed genes of select subsets in days 4 and 5 of PSP
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uteri were validated by Real-time PCR as described [10]. Total RNA was
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extracted
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(Invitrogen-15596018, Carlsbad, CA) according to the manufacturer’s protocol.
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A total of 3 µg RNA was used to synthesize cDNA with the oligo dT primers.
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Quantitative Real-time PCR was performed with SYBR Green dye method
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(Takara, DRR820A) on an ABI PRISM 7500 system. All expression values
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were normalized against Gapdh. The primer sequences for Real-time PCR
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were listed in Supplementary Table 1. All Real-time PCR experiments were
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repeated at least 3 times.
days
4
and
5
PSP
uteri
using
Trizol
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2.7 Statistical analysis
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Statistical analysis was performed with the SPSS 11.5 program (SPSS Inc.,
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Chicago, IL, USA) for embryo transfers and validation experiment data.
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Comparison
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independent-samples Student t-test. The data are shown as means ± SD., P
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values of statistical significance are represented as ***P < 0.001, **P < 0.01,
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*P<0.05.
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3. Result
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3.1 Uterine status beyond day 5 of pregnancy was unfavorable for blastocyst
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implantation in mice
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In this study, first we comprehensively analyzed the implantation status
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beyond normal "window" of uterine receptivity, which spanned from day 4 to
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day 6 of PSP. Embryo transfer experiments were conducted on various days of
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PSP and implantation status was examined at indicted time points. As shown
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in Figure 1A and Table1, when blastocysts were transferred into the day 4
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recipients, normal embryo implantation occurred in all the recipients 24 hours
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after the transfer, but a considerable portion (3 out of 8 mice) of day 5 PSP
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recipients exhibited no implantation sites examined 24 hours later. Interestingly,
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almost all day 5 PSP recipients displayed implantation sites when examined
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48 hours after the transfer. However, blastocysts transferred to recipients on
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day 6 of PSP almost completely failed to implant even examined 72 hours
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later,(Figure 1A & Table 1). Nonetheless, blastocysts transferred into day 5
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PSP recipients showed considerably reduced rates of implantation sites
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(38.9% vs 77.9%) compared with day 4 recipients detected 48 hours later
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(Figure 1A & Table 1).
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3.2 Embryos implanting beyond the normal window exhibited abnormal
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mid-gestational development
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These
peri-implantation
defects
prompted
us
to
scrutinize
post-implantation embryo development in these day 5 PSP recipients. We
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examined the pregnancy status on day 12 of pregnancy or 8 days after the
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transfer, which represented the mid-gestation phase. As shown in Figure 1B,
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while most of the implantation sites in day 4 recipients were well spaced along
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the uterine horns and developed normally on day 12, a large portion of
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implantation sites in day 5 recipient mice were smaller or appeared altered
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positioning/crowding, and even showed signs of resorption.
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3.3 Deferred implantation beyond the normal window compromised term
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pregnancy success
Next, we also examined the fertility of these transferred recipients. The
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successful pregnancy rate was significantly reduced in day 5 PSP recipient
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mice compared with those in day 4, as nearly half of the day 5 PSP recipient
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mice did not produce any pups. In addition, the percentage of pups delivered
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at term was markedly lower in the day 5 PSP recipient mice (n=117, 48.72%
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versus n=108, 12.96%) (Figure 1C).
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3.4 Uteri on days 5-6 showed impaired artificial decidualization Given that the decidualization process could occur only in the receptive
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uterus, we utilized the artificial decidualization model to evaluate the state of
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uterine receptivity on different days of pregnancy. As shown in Figure 1D,
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artificially induced decidualization displayed by the deciduoma formation
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occurred in the uteri of days 4 and 5 PSP, but never in the uteri of day 6 PSP,
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which is consistent with the embryo transfer data. Though the decidual
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responses were observed in day 5 PSP mice, the fold increases of uterine
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weight (Figure 1E), which is a parameter for decidual differentiation extent,
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was lower than that of day 4 uteri.
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3.5 Microarray analysis revealed differential uterine gene expression profile on
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day 4 versus day 5 of PSP
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In order to uncover the molecular differences between these two
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physiological different states of uterine receptivity on day 4 and day 5, the
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microarray approach was utilized to analyze the differentially expressed genes.
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A complete gene expression data set, representing about 24, 000 gene
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features, is available at the Affymetrix GeneChip mouse Genome Array.
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approximately 229 differentially expressed genes were found and selected for
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further functional analysis (Figure 2A). Among them, 111 genes have higher
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expression in day 4 PSP uteri, whereas the remaining 118 genes are
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prominent in day 5 PSP uteri (Figure 2B and C). GO functional classification
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analysis of the 229 differentially expressed genes revealed that 30 genes were
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related to proteolysis and signal transduction, and others fell into various other
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biological processes such as oxidation reduction, cytolysis, inflammatory and
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immune response etc. (Figure 2D & Table 2). In the signal transduction
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classification, some molecules, such as Lpar3 and Lgr5, have been proven to
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be critical for normal embryo implantation and decidualization [14, 15].
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3.6 Epithelium-expressing genes showed dramatic differences in day 4 versus
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day 5 pseudopregnant uteri
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As the uterine epithelium firstly interacted with the blastocyst and a
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dynamic gene expression program is involved during the uterine epithelium
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differentiation for the establishment of uterine receptivity, we further intend to
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explore the molecular defects of epithelium differentiation beyond the normal
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implantation “window”. For this purpose, we compared our data to the publicly
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available data about the uterine luminal epithelium-expressing (ULE) genes
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[referred the microarray data (GEO number: GSE44451)] [16]. As shown in
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Figure 3A, the diagram showed the overlap between 229 differentially
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expressed genes and day 4 ULE genes. The results identified a large
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proportion
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implantation-related ULE genes. Moreover, functional classification analysis of
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the 107 different ULE genes showed enrichment in transport, P4 response,
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lipid metabolic process and oxidation-reduction process etc. (Figure 3B). For
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example, as shown in Figure 3C-F, the epithelial specifically expressed genes
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Sprr2f, Foxa-2 and Lpar3, were significantly down-regulated in day 5 PSP uteri,
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whereas Cyp26a1 were notably up-regulated compared with day 4 PSP uteri.
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Some of these genes have been proven to be indispensable for implantation
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[14, 17, 18].
differentially
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of
3.7 Progesterone-responsive genes were aberrantly expressed in uterine
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epithelium on day 5 of PSP
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As stated above, the P4 response was a significant category for the
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differentially expressed genes between the day 4 and day 5 PSP uteri.
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Real-time PCR analyses confirmed the epithelium specific expressing genes,
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which also responded to P4 signal, such as Fxyd4, Jam-2, Lrp-2, Areg, Galb1
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and Sox-17 were down-regulated in day 5 uteri (Figure 4A-F). In addition, 15
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responsiveness to P4, did not display significant changes (Figure 4G-H),
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indicating that uterine receptivity defects in day 5 PSP recipients could be
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mainly related to defective epithelial P4 signaling.
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Embryo implantation involves the intimate interaction between an
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implantation-competent blastocyst and a receptive uterus, which occurs in a
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limited time period known as the “window” of implantation [8]. Molecular and
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genetic evidence indicates that diverse signaling molecules produced by the
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epithelial and stromal cells, including cytokines, lipid molecule, homeobox
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transcription factors, and morphogen genes, function in the autocrine,
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paracrine and juxtacrine manner to regulate the complex process of
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implantation [7, 8]. Normal embryo implantation is critical for the establishment
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of a successful pregnancy. Defective implantation processes can cause
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adverse “ripple effects”, leading to abnormal decidualization and placentation,
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retarded fetal development, and poor pregnancy outcomes, even infertility [7].
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In human, it was also observed that an increased incidence of early pregnancy
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loss was related with the implantation occurred outside the prescribed window
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of uterine receptivity [19].
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In this study, we found that the uterine receptivity had severe defects on
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day 5 in mice; though the blastocysts could still implant in the day 5 uteri with
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longer time to initiate the attachment reaction, the implantation rate and the
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growth and development of the embryos during the post-Implantation stage,
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were both severely influenced in the day 5 transfer group compared with that 17
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of day 4. The "window" to initiate attachment reaction was almost shut off on day 6
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of pregnancy in mice, which was consistent with the previous report [11]. The
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abnormal uterine receptivity on day 5 and day 6 was further demonstrated by
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the defective response to the artificial decidualization stimuli. The findings
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demonstrated that though mouse uteri could partially accept the blastocyst to
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initiate the implantation process on day 5 of PSP, the day 5 uteri were
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somehow defective for implantation as compared with the normal day 4
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receptive uteri and the normal "window" for implantation is restricted to a
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limited time span which is required for successful pregnancy outcome.
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Then, we performed integral microarray analysis of day 4 and 5 PSP uteri
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to explore the molecular difference that distinguished the normal uterine
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receptivity from the defective one. Our findings herein provided a global view
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of the differentially regulated cellular and molecular processes, including
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transport, metabolism, proteolysis, signal transduction, oxidation reduction,
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and inflammatory response in day 4 versus day 5 PSP uteri.
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In particular, we revealed that abundant epithelium-expressing genes had
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dramatic changes beyond the normal "window" of implantation, highlighting the
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physiological significance of normal differentiated epithelium for the
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establishment and maintenance of uterine receptivity. It also suggested that 18
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the underlying molecular cause for the defective uterine receptivity on day 5 of
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PSP. The Foxa2 member has been shown to be expressed specifically in the
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glandular epithelium of the murine uterus. Conditionally knockout Foxa2 in the
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mouse uterus showed significantly reduced fertility due to disrupted blastocyst
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implantation and impaired decidual response [17]. Endometrial gland
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secretions play vital biological roles in regulating uterine receptivity and
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stromal cell decidualization [20, 21]. Indeed, deficient glandular activity
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described as a secretory-phase defect is linked to early pregnancy failure in
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humans [22]. Lpar3, which is the LPA receptor and specifically expressed in
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the luminal epithelium during the peri-implantation stage, was significantly
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down-regulated (Figure 3E). Targeted deletion of Lpar3 in mice resulted in
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significantly reduced litter size, which could be attributed to deferred
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implantation and altered embryo spacing [14]. Cytochrome P450 26A1
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(Cyp26a1), a retinoic acid (RA)-metabolizing enzyme involved in Vitamin A (VA)
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metabolism, specifically expressed in the uterine luminal epithelium, is
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important for blastocyst implantation. However, excessive VA intake during
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pregnancy may also lead to adverse maternal and fetal effects [18]. In our data
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it was significantly up-regulated in day 5 PSP uteri, which might lead to an
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adverse Vitamin A microenvironment in the uteri for embryo implantation
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(Figure 3F). Many of the differentially expressed genes in epithelium on day 5
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that the P4 supplement could partially retain uterine receptivity for blastocyst
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implantation and decidualization on day 6 of PSP. So our result that the
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compromised P4 response in day 5 of PSP is consistent with the previous
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report. It also suggested that the normal P4 response, especially the epithelial
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P4-PR signaling was critical for the normal uterine receptivity [11]. However,
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this may not be influenced by PR protein level since PR still remained highly
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expressed in the day 5 pseudopregnant LE and the molecular basis need
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further study [23].
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The study presents more opportunities to select candidate genes involved
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in optimizing uterine environment for implantation. Significantly, these results
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provide evidence that two different physiological states of uterine receptivity
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are molecularly distinguishable in a global perspective and underscore the
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significant changes of epithelial expressed molecular pathways in these
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processes, especially the compromised epithelial P4 pathway rather than
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stromal. Beyond that, our findings provide valuable clinical reference that a
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better understanding of the uterine receptivity could advance discovery of
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novel biomarkers of uterine receptivity for diagnosing and treating infertility in
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human IVF-ET programs.
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We are grateful to lab members Drs. Wengeng Lu and Zhaowei Tu for embryo
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transfer assistance. This work was supported by the National Key R&D Plan
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and National Basic Research Program of China (2017YFA0104603, 81601285
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to S.K.; 81130009, 81330017 and 81490744 to H. W.).
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[9] Mulac-Jericevic B, Mullinax RA, DeMayo FJ, Lydon JP, Conneely OM. Subgroup of reproductive functions of progesterone mediated by progesterone receptor-B isoform. Science. 2000;289:1751-4. [10] Zhang S, Kong S, Wang B, Cheng X, Chen Y, Wu W, et al. Uterine Rbpj is required for
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embryonic-uterine orientation and decidual remodeling via Notch pathway-independent and -dependent mechanisms. Cell Res. 2014;24:925-42. [11] Song H, Han K, Lim H. Progesterone supplementation extends uterine receptivity for blastocyst implantation in mice. Reproduction. 2007;133:487-93. [12] Wang Q, Lu J, Zhang S, Wang S, Wang W, Wang B, et al. Wnt6 is essential for stromal cell
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proliferation during decidualization in mice. Biol Reprod. 2013;88:5. [13] Huang da W, Sherman BT, Lempicki RA. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc. 2009;4:44-57.
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[14] Ye X, Hama K, Contos JJ, Anliker B, Inoue A, Skinner MK, et al. LPA3-mediated lysophosphatidic acid signalling in embryo implantation and spacing. Nature. 2005;435:104-8. [15] Sun X, Terakawa J, Clevers H, Barker N, Daikoku T, Dey SK. Ovarian LGR5 is critical for successful pregnancy. FASEB J. 2014;28:2380-9. [16] Xiao S, Diao H, Zhao F, Li R, He N, Ye X. Differential gene expression profiling of mouse uterine luminal epithelium during periimplantation. Reprod Sci. 2014;21:351-62. [17] Jeong JW, Kwak I, Lee KY, Kim TH, Large MJ, Stewart CL, et al. Foxa2 is essential for mouse endometrial gland development and fertility. Biol Reprod. 2010;83:396-403. [18] Han BC, Xia HF, Sun J, Yang Y, Peng JP. Retinoic acid-metabolizing enzyme cytochrome P450 26a1 (cyp26a1) is essential for implantation: functional study of its role in early pregnancy. J Cell Physiol.
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ACCEPTED MANUSCRIPT 2010;223:471-9. [19] Wilcox AJ, Baird DD, Weinberg CR. Time of implantation of the conceptus and loss of pregnancy. N Engl J Med. 1999;340:1796-9. [20] Cooke PS, Spencer TE, Bartol FF, Hayashi K. Uterine glands: development, function and experimental model systems. Mol Hum Reprod. 2013;19:547-58. [21] Filant J, Spencer TE. Endometrial glands are essential for blastocyst implantation and decidualization in the mouse uterus. Biol Reprod. 2013;88:93.
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[22] Filant J, Spencer TE. Cell-specific transcriptional profiling reveals candidate mechanisms regulating development and function of uterine epithelia in mice. Biol Reprod. 2013;89:86.
[23] Diao H, Paria BC, Xiao S, Ye X. Temporal expression pattern of progesterone receptor in the uterine luminal epithelium suggests its requirement during early events of implantation. Fertil Steril. 2011;95:2087-93.
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Figure Legends:
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Figure1 Uterine receptivity and decidualization were defective beyond
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normal implantation "window"
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(A) Implantation status of blastocyst transferred on indicted days of PSP. (B)
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Post-implantation development of embryos transferred on indicted days of PSP.
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(C) The pregnancy outcome of day 4 blastocysts transferred into recipients on
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days 4 or 5 of PSP. Gross morphology (D) and weight induction (E) of uteri in
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response to artificial induction of decidualization in days 4, 5 and 6 PSP
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recipients. Data are shown as means ± SD, (*P<0.05, ***P<0.001).
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Figure2 Clustering analyses identified differentially expressed genes in
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days 4 and 5 PSP uteri
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(A) A volcano plot of gene expression in days 4 and 5 PSP uteri. (B) The Venn
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diagram showed the summary of total identified genes in days 4 and 5 uteri. (C)
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The heatmap of different expressed genes in each experimental group. The
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1-3 represented three independent experiments respectively. (D) Gene
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Ontology analysis of the 229 differentially expressed genes.
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and 5 PSP uteri
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(A) The diagram showed the 107 different expressed ULE genes in the total
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229 differentially expressed genes. (B) Gene Ontology analysis of the 107
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different ULE genes. (C-F) mRNA expression level of Sprr2f, Foxa-2, Lpar3
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and Cyp26a1, were determined in days 4, 5 PSP uteri by quantitative
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Real-time PCR respectively. Values are expressed as mean ± SD from at least
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three independent samples (*P<0.05, **P<0.01).
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Figure4 Differential expression of epithelium specific P4-responsive
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genes in days 4 and 5 PSP uteri
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(A-H) Expression level of epithelial specific Fxyd4, Jam-2, Lrp-2, Areg, Galb1,
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Sox-17 genes and stromal special Hand-2, Hoxa-10 P4-response genes was
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determined in days 4, 5 PSP uteri by quantitative Real-time PCR, respectively.
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Values are expressed as mean ± SD from at least three independent samples
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(*P<0.05).
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ACCEPTED MANUSCRIPT Table 1. Implantation of blastocysts transferred into pseudopregnant mice No. of recipients with IS(%) 10(100) 8(100) 7(100)
No. of IS (%)
10 8 7
No. of embryos transferred 121 104 112
Day 5
24h 48h 72h
8 7 7
104 90 92
6(62.5) 7(100) 6(85.7)
31(29.8)* 35(38.9)* 36(39.1)*
Day 6
24h 48h 72h
5 5 5
69 66 74
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82(67.8) 81(77.9) 88(78.6)
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No. of recipients
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Day 4
Times after transfer 24h 48h 72h
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2(40) 1(20) 0
5(7.3) * 2(3.0) * 0*
ACCEPTED MANUSCRIPT Table 2. Functional categories of selected genes differentially expressed in day4 and day5 recipients according to gene ontology and KEGG pathway analyses Gene Symbol
Fold change
q-value(%)
Inflammatory response
Cxcl15 Kng1 Ccr2 Ccl21c Ccl21b C3 Pla2g7 Ccl8
4.09 0.26 0.48 0.42 0.42 0.42 0.47 0.45
0 0 0 0.3 0.3 0.55 0.55 0.9
Signal transduction
Tnfrsf21 Pex11a Iqgap2 Lpar3 Lgr5 Fga Arnt2 Ccr2 Ptger2 Tnfrsf11b Ms4a4c
2.02 2.16 2.00 2.15 0.11 0.05 0.38 0.48 0.31 0.46 0.28
0 0 0.23 0.43 0 0 0 0 0 0.61 0.74
Cxcl15 Pf4 Ccl21c Ccl21b Ccl8
4.09 0.44 0.43 0.43 0.46
0 0 0.30 0.30 0.99
Adh7 Mthfd2 Chdh Cdo1 Cbr2 Srd5a3 Cyp26a1 Me1 Xdh Gpx2
3.23 2.49 2.01 2.06 3.07 3.35 0.07 0.49 0.49 0.46
0 0 0 0 0 0.41 0 0 0.55 0.55
Oxidation reduction
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Functional category
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ACCEPTED MANUSCRIPT The receptive state of the uterus is defined as a limited time period when the uterine milieu is favorable for blastocyst implantation. It is generally considered as day 4 of pregnancy in mice. In this study, by utilizing the embryo transfer
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model, we observed that a portion of blastocysts can initiate implantation even when transferred in day 5, while their mid-gestational development exhibited severe retardation, proving that the uterine status beyond the normal
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implantation window is unconducive for implantation. We therefore performed
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microarray analysis to explore the molecular basis that distinguishes the normal and defective uterine receptivity. Approximate 229 genes were found to be
differentially
expressed,
among
which
a
large
amount
were
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epithelial-expressing genes and those responsive to progesterone signaling.
S0093-691X(17)30390-4
DOI:
10.1016/j.theriogenology.2017.08.005
Reference:
THE 14225
To appear in:
Theriogenology
Received Date: 11 November 2016 Revised Date:
3 August 2017
Accepted Date: 3 August 2017
Please cite this article as: Xin Q, Li M, Wang H, Cui S, Xia G, Kong S, Transcript analysis identifies differential uterine gene expression profile beyond the normal implantation window in mice, Theriogenology (2017), doi: 10.1016/j.theriogenology.2017.08.005. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Revised
ACCEPTED MANUSCRIPT Transcript analysis identifies differential uterine gene expression profile
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beyond the normal implantation window in mice
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Qiliang Xina,1, Meiling Lia,1, Haibin Wangb, Sheng Cuia, Guoliang Xiaa,*,
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Shuangbo Kongb,*
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a
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China Agricultural University, Beijing 100193, PR China;
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b
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University, Medical College Xiamen University, Xiamen, Fujian 361102, PR
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China.
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State Key Laboratory of Agro-biotechnology, College of Biological Sciences,
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Reproductive Medical Center, The First Affiliated Hospital of Xiamen
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Authors contributed equally to this work.
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Correspondence: Shuangbo Kong (Email: [email protected]) or
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*
Guoliang Xia (Email: [email protected])
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Running title: Uterine gene expression beyond implantation window
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Competing Interest: The authors declare no conflict of interest. 1
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Abstract Uterine receptivity is defined as a state when the uterine milieu is favorable
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for blastocyst implantation and it can only last for a limited time period. In this
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study, by utilizing the embryo transfer model, it was observed that a portion of
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blastocysts could initiate implantation even when transferred beyond the timing
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of normal uterine receptivity, while their mid-gestational embryo development
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exhibited severe retardation, suggesting that the uterine status beyond the
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normal implantation window is unconducive for normal implantation. We
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further performed microarray analysis to explore the molecular basis that
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distinguishes the normal and defective uterine receptivity. A total of 229 genes
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was found to be differentially expressed, and a large amount of them were
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epithelium-expressing genes and responsive to progesterone signaling.
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Keywords:
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progesterone-responsive genes
receptivity;
microarray;
epithelium;
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uterine
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Abbreviations: LE, Luminal epithelium; PSP, Pseudo-pregnancy; P4,
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Progesterone; E2, Estrogen; IS, Implantation site; Cyp26a1, Cytochrome P450
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26A1; PR, Progesterone receptor.
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1. Introduction Embryo implantation is one of the crucial steps in mammalian embryo
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development. In placental mammals, uterine sensitivity to implantation is
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classified into pre-receptive, receptive and non-receptive (refractory) phases
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[1-3]. In normal pregnancy, when implantation-competent blastocysts are
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ready to initiate implantation, the uterus differentiates into an altered state
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called uterine receptivity, which sustained for a limited time period. In mice, the
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normal uterine receptive phase is on day 4 of pregnancy or pseudo-pregnancy
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(day 1 = day of vaginal plug). At this stage, the uterine environment is
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conducive to blastocyst growth, attachment and subsequent events of
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implantation. In contrast, the uterine environment is unfavorable to blastocyst
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survival in the refractory phase after day 5 of pregnancy [1, 4]. Embryo
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implantation is a dynamic developmental event that involves a series of
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physical and physiological interactions between the blastocyst trophectoderm
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and various endometrial cell-types, including both the uterine epithelial and
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stromal compartment. It has been observed that the ultrastructure of luminal
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epithelium (LE), such as LE cell surface components, lateral adherent
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junctions and gap junction channels, need remolding during the establishment
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of uterine receptivity. The morphological transformations are likely associated
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with the differential gene expression in the LE, which would affect the
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ACCEPTED MANUSCRIPT trophectoderm-uterine epithelium crosstalk that fosters the normal blastocyst
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implantation. In response to the implanting embryo, the surrounding uterine
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stroma undergoes extensive cellular proliferation and transformation, a
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process known as decidualization, to accommodate embryonic growth and
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invasion. Decidualization can also occur in response to artificial stimulus by oil
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injection or endometrium scratch in the receptive uterus to form deciduoma.
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In mice, progesterone (P4), coordinating with estrogen (E2), is obligatory
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for uterine preparation for a receptive state [1, 5-7]. In mice, there are two main
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P4 receptor isoforms as PRA and PRB, arising from alternative promoter
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usage in the same gene Pgr [8]. The P4 mainly functions through its nuclear
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receptor PRA to coordinate the molecular networks for the establishment of
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uterine receptivity in mice. Numerous defects in female mice lacking the Pgr
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gene that encodes PR have been demonstrated, including failure in ovulation,
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mammary gland development, and embryo implantation along with uterine
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hyperplasia and inflammation, reflecting the critical role of P4/PR pathway in
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female reproduction [9]. Furthermore, the molecular and genetic evidence has
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also indicated that locally produced signaling molecules in the uterus, including
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cytokines, homeobox transcription factors, coordinate with these hormone
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receptors and serve as autocrine, paracrine and juxtacrine factors to specify
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the uterine receptivity [8].
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portion of blastocysts can initiate implantation even when transferred on day 5
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beyond the normal timing of uterine receptivity, while the mid-gestational
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development of these embryos exhibited severe retardation, suggesting that
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the uterine status on day 5 of pseudopregnancy (PSP) is unconducive for
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normal implantation. So we inferred that critical physiological changes occur in
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the uteri between days 4 and 5 of PSP. In order to explore the underlying
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molecular differences between these two different uterine states, we applied
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the microarray approach to analyze the uterine gene expression profile on day
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4 versus day 5 of PSP. Using this approach, here we show that, 229 out of
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about 24, 000 genes examined were significantly differentially expressed
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between days 4 and 5 PSP uteri. Further analyses revealed that a large
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proportion of (46.7%) differentially expressed genes were uterine epithelium-
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expressing genes and responsive to P4 signaling, which may compromise the
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epithelial cell differentiation program and thus the normal implantation.
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2. Materials and Methods:
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2.1 Mice
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Adult 8 weeks CD-1 male and female mice used in this study were
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purchased from the Vital River Laboratory Animal Technology Co. Ltd. All
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animal experiments were performed in accordance with the guidelines and
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regulations of the Institutional Animal Care and Use Committee of China
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Agricultural University. Adult female mice were mated with intact or
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vasectomized males of the same strain to induce pregnancy or PSP,
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respectively. The morning of seeing a vaginal plug was defined as day 1 of
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pregnancy or PSP.
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2.2 Embryo Transfer and examination of pregnancy outcome
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Day 4 blastocysts from the normal pregnant female donors were
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transferred to the uteri of recipients in the morning (10:00 h) on days 4–6 of
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PSP. Recipient mice were sacrificed to examine the implantation status at
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10:00 h on indicated days. The number of implantation site (IS) was recorded
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by tail vein injection (0.1 ml/mouse) of Chicago Blue dye (Sigma-Aldrich,
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C8679) solution (1% in saline) at 48 hours after embryo transfer. Mice were
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the uterine horns since the vascular permeability is increased at the
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implantation sites [10, 11]. In the mice without implantation sites, the uterine
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horns were flushed with saline to recover the unimplanted blastocysts. Mice
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without implantation sites but no recovery of blastocysts were excluded from
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the experiments. Recipients were examined for subsequent developmental
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events on day 12 or observed for delivery of pups at term. For each time point,
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at least 5 mice were included for the analysis.
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2.3 Experimentally induced decidualization in pseudo-pregnant mice To induce artificial decidualization, 25 µl of sesame oil (Sigma-S3547) was
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infused intraluminally in one uterine horn on days 4–6 of PSP. The
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contra-lateral horn served as control. At least 4 mice were examined in each
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time point for this experiment. Mice were sacrificed 96 hours later. Uterine
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weights of the infused and non-infused (control) horns were recorded. Fold
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increases in uterine weights were used as an index of decidualization [12].
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2.4 RNA extraction, amplification, labeling and hybridization For days 4 and 5 PSP mouse uteri (n=4 mice per group), the samples 7
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reagent (Invitrogen-15596018, Carlsbad, CA) according to manufacturer’s
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instructions. The RNA was cleaned up with RNeasy Kit (Cat #74104, Qiagen,
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Hilden, Germany) and the quantities and qualities were determined by
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spectrophotometer and 1% formaldehyde denaturing gel electrophoresis.
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Affymetrix GeneChip mouse Genome Array, which contains more than 45,000
sets
representing
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characterized mouse genes, was used in microarray analysis. Hybridization,
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data capture, and analysis were performed by Capital Bio Corporation (Beijing,
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China), a service provider authorized by Affymetrix Inc. (Santa Clara, CA).
24,000
of
the
best
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approximately
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2.5 Microarray Data Analysis
All the data were viewed pre-normalized by tree clustering to remove
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outlying microarray data, leaving three arrays (three replicates) per time point.
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The hybridization data were analyzed using Affymetrix GeneChip
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Command Console Software (AGCC), which uses statistical criteria to
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generate a ‘present’ or ‘absent’ call for genes represented by each probe set
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on the array. Afterwards, genes with ‘absent’ scores were filtered out and the
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remaining genes were analyzed. Microarray data were normalized using the
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package (http://www.bioconductor.org) was used to calculate expression
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values, which refer to the quantile normalization of Robust Multiarray Average
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method (each performed at the individual probe level). Significance Analysis of
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Microarrays (SAM) was used to identify genes that are differentially expressed.
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[13].
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2.6 Validation of microarray data by Real-time PCR
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Differentially expressed genes of select subsets in days 4 and 5 of PSP
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uteri were validated by Real-time PCR as described [10]. Total RNA was
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extracted
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(Invitrogen-15596018, Carlsbad, CA) according to the manufacturer’s protocol.
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A total of 3 µg RNA was used to synthesize cDNA with the oligo dT primers.
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Quantitative Real-time PCR was performed with SYBR Green dye method
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(Takara, DRR820A) on an ABI PRISM 7500 system. All expression values
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were normalized against Gapdh. The primer sequences for Real-time PCR
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were listed in Supplementary Table 1. All Real-time PCR experiments were
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repeated at least 3 times.
days
4
and
5
PSP
uteri
using
Trizol
reagent
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2.7 Statistical analysis
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Statistical analysis was performed with the SPSS 11.5 program (SPSS Inc.,
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Chicago, IL, USA) for embryo transfers and validation experiment data.
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Comparison
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independent-samples Student t-test. The data are shown as means ± SD., P
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values of statistical significance are represented as ***P < 0.001, **P < 0.01,
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*P<0.05.
was
presented
by
making
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3. Result
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3.1 Uterine status beyond day 5 of pregnancy was unfavorable for blastocyst
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implantation in mice
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In this study, first we comprehensively analyzed the implantation status
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beyond normal "window" of uterine receptivity, which spanned from day 4 to
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day 6 of PSP. Embryo transfer experiments were conducted on various days of
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PSP and implantation status was examined at indicted time points. As shown
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in Figure 1A and Table1, when blastocysts were transferred into the day 4
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recipients, normal embryo implantation occurred in all the recipients 24 hours
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after the transfer, but a considerable portion (3 out of 8 mice) of day 5 PSP
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recipients exhibited no implantation sites examined 24 hours later. Interestingly,
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almost all day 5 PSP recipients displayed implantation sites when examined
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48 hours after the transfer. However, blastocysts transferred to recipients on
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day 6 of PSP almost completely failed to implant even examined 72 hours
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later,(Figure 1A & Table 1). Nonetheless, blastocysts transferred into day 5
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PSP recipients showed considerably reduced rates of implantation sites
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(38.9% vs 77.9%) compared with day 4 recipients detected 48 hours later
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(Figure 1A & Table 1).
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3.2 Embryos implanting beyond the normal window exhibited abnormal
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mid-gestational development
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These
peri-implantation
defects
prompted
us
to
scrutinize
post-implantation embryo development in these day 5 PSP recipients. We
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examined the pregnancy status on day 12 of pregnancy or 8 days after the
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transfer, which represented the mid-gestation phase. As shown in Figure 1B,
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while most of the implantation sites in day 4 recipients were well spaced along
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the uterine horns and developed normally on day 12, a large portion of
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implantation sites in day 5 recipient mice were smaller or appeared altered
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positioning/crowding, and even showed signs of resorption.
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3.3 Deferred implantation beyond the normal window compromised term
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pregnancy success
Next, we also examined the fertility of these transferred recipients. The
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successful pregnancy rate was significantly reduced in day 5 PSP recipient
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mice compared with those in day 4, as nearly half of the day 5 PSP recipient
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mice did not produce any pups. In addition, the percentage of pups delivered
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at term was markedly lower in the day 5 PSP recipient mice (n=117, 48.72%
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versus n=108, 12.96%) (Figure 1C).
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3.4 Uteri on days 5-6 showed impaired artificial decidualization Given that the decidualization process could occur only in the receptive
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uterus, we utilized the artificial decidualization model to evaluate the state of
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uterine receptivity on different days of pregnancy. As shown in Figure 1D,
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artificially induced decidualization displayed by the deciduoma formation
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occurred in the uteri of days 4 and 5 PSP, but never in the uteri of day 6 PSP,
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which is consistent with the embryo transfer data. Though the decidual
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responses were observed in day 5 PSP mice, the fold increases of uterine
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weight (Figure 1E), which is a parameter for decidual differentiation extent,
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was lower than that of day 4 uteri.
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3.5 Microarray analysis revealed differential uterine gene expression profile on
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day 4 versus day 5 of PSP
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In order to uncover the molecular differences between these two
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physiological different states of uterine receptivity on day 4 and day 5, the
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microarray approach was utilized to analyze the differentially expressed genes.
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A complete gene expression data set, representing about 24, 000 gene
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features, is available at the Affymetrix GeneChip mouse Genome Array.
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approximately 229 differentially expressed genes were found and selected for
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further functional analysis (Figure 2A). Among them, 111 genes have higher
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expression in day 4 PSP uteri, whereas the remaining 118 genes are
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prominent in day 5 PSP uteri (Figure 2B and C). GO functional classification
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analysis of the 229 differentially expressed genes revealed that 30 genes were
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related to proteolysis and signal transduction, and others fell into various other
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biological processes such as oxidation reduction, cytolysis, inflammatory and
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immune response etc. (Figure 2D & Table 2). In the signal transduction
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classification, some molecules, such as Lpar3 and Lgr5, have been proven to
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be critical for normal embryo implantation and decidualization [14, 15].
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3.6 Epithelium-expressing genes showed dramatic differences in day 4 versus
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day 5 pseudopregnant uteri
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As the uterine epithelium firstly interacted with the blastocyst and a
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dynamic gene expression program is involved during the uterine epithelium
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differentiation for the establishment of uterine receptivity, we further intend to
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explore the molecular defects of epithelium differentiation beyond the normal
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implantation “window”. For this purpose, we compared our data to the publicly
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available data about the uterine luminal epithelium-expressing (ULE) genes
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[referred the microarray data (GEO number: GSE44451)] [16]. As shown in
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Figure 3A, the diagram showed the overlap between 229 differentially
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expressed genes and day 4 ULE genes. The results identified a large
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proportion
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implantation-related ULE genes. Moreover, functional classification analysis of
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the 107 different ULE genes showed enrichment in transport, P4 response,
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lipid metabolic process and oxidation-reduction process etc. (Figure 3B). For
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example, as shown in Figure 3C-F, the epithelial specifically expressed genes
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Sprr2f, Foxa-2 and Lpar3, were significantly down-regulated in day 5 PSP uteri,
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whereas Cyp26a1 were notably up-regulated compared with day 4 PSP uteri.
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Some of these genes have been proven to be indispensable for implantation
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[14, 17, 18].
differentially
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of
3.7 Progesterone-responsive genes were aberrantly expressed in uterine
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epithelium on day 5 of PSP
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As stated above, the P4 response was a significant category for the
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differentially expressed genes between the day 4 and day 5 PSP uteri.
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Real-time PCR analyses confirmed the epithelium specific expressing genes,
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which also responded to P4 signal, such as Fxyd4, Jam-2, Lrp-2, Areg, Galb1
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and Sox-17 were down-regulated in day 5 uteri (Figure 4A-F). In addition, 15
ACCEPTED MANUSCRIPT Hand-2 and HoxA-10, two stromal cell expressed genes for uterine
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responsiveness to P4, did not display significant changes (Figure 4G-H),
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indicating that uterine receptivity defects in day 5 PSP recipients could be
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mainly related to defective epithelial P4 signaling.
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Embryo implantation involves the intimate interaction between an
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implantation-competent blastocyst and a receptive uterus, which occurs in a
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limited time period known as the “window” of implantation [8]. Molecular and
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genetic evidence indicates that diverse signaling molecules produced by the
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epithelial and stromal cells, including cytokines, lipid molecule, homeobox
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transcription factors, and morphogen genes, function in the autocrine,
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paracrine and juxtacrine manner to regulate the complex process of
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implantation [7, 8]. Normal embryo implantation is critical for the establishment
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of a successful pregnancy. Defective implantation processes can cause
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adverse “ripple effects”, leading to abnormal decidualization and placentation,
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retarded fetal development, and poor pregnancy outcomes, even infertility [7].
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In human, it was also observed that an increased incidence of early pregnancy
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loss was related with the implantation occurred outside the prescribed window
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of uterine receptivity [19].
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In this study, we found that the uterine receptivity had severe defects on
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day 5 in mice; though the blastocysts could still implant in the day 5 uteri with
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longer time to initiate the attachment reaction, the implantation rate and the
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growth and development of the embryos during the post-Implantation stage,
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were both severely influenced in the day 5 transfer group compared with that 17
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of day 4. The "window" to initiate attachment reaction was almost shut off on day 6
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of pregnancy in mice, which was consistent with the previous report [11]. The
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abnormal uterine receptivity on day 5 and day 6 was further demonstrated by
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the defective response to the artificial decidualization stimuli. The findings
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demonstrated that though mouse uteri could partially accept the blastocyst to
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initiate the implantation process on day 5 of PSP, the day 5 uteri were
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somehow defective for implantation as compared with the normal day 4
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receptive uteri and the normal "window" for implantation is restricted to a
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limited time span which is required for successful pregnancy outcome.
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Then, we performed integral microarray analysis of day 4 and 5 PSP uteri
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to explore the molecular difference that distinguished the normal uterine
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receptivity from the defective one. Our findings herein provided a global view
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of the differentially regulated cellular and molecular processes, including
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transport, metabolism, proteolysis, signal transduction, oxidation reduction,
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and inflammatory response in day 4 versus day 5 PSP uteri.
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In particular, we revealed that abundant epithelium-expressing genes had
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dramatic changes beyond the normal "window" of implantation, highlighting the
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physiological significance of normal differentiated epithelium for the
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establishment and maintenance of uterine receptivity. It also suggested that 18
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the underlying molecular cause for the defective uterine receptivity on day 5 of
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PSP. The Foxa2 member has been shown to be expressed specifically in the
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glandular epithelium of the murine uterus. Conditionally knockout Foxa2 in the
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mouse uterus showed significantly reduced fertility due to disrupted blastocyst
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implantation and impaired decidual response [17]. Endometrial gland
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secretions play vital biological roles in regulating uterine receptivity and
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stromal cell decidualization [20, 21]. Indeed, deficient glandular activity
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described as a secretory-phase defect is linked to early pregnancy failure in
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humans [22]. Lpar3, which is the LPA receptor and specifically expressed in
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the luminal epithelium during the peri-implantation stage, was significantly
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down-regulated (Figure 3E). Targeted deletion of Lpar3 in mice resulted in
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significantly reduced litter size, which could be attributed to deferred
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implantation and altered embryo spacing [14]. Cytochrome P450 26A1
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(Cyp26a1), a retinoic acid (RA)-metabolizing enzyme involved in Vitamin A (VA)
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metabolism, specifically expressed in the uterine luminal epithelium, is
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important for blastocyst implantation. However, excessive VA intake during
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pregnancy may also lead to adverse maternal and fetal effects [18]. In our data
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it was significantly up-regulated in day 5 PSP uteri, which might lead to an
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adverse Vitamin A microenvironment in the uteri for embryo implantation
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(Figure 3F). Many of the differentially expressed genes in epithelium on day 5
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ACCEPTED MANUSCRIPT of PSP were indeed responded to P4 signal, and previous study have proven
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that the P4 supplement could partially retain uterine receptivity for blastocyst
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implantation and decidualization on day 6 of PSP. So our result that the
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compromised P4 response in day 5 of PSP is consistent with the previous
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report. It also suggested that the normal P4 response, especially the epithelial
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P4-PR signaling was critical for the normal uterine receptivity [11]. However,
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this may not be influenced by PR protein level since PR still remained highly
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expressed in the day 5 pseudopregnant LE and the molecular basis need
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further study [23].
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The study presents more opportunities to select candidate genes involved
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in optimizing uterine environment for implantation. Significantly, these results
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provide evidence that two different physiological states of uterine receptivity
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are molecularly distinguishable in a global perspective and underscore the
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significant changes of epithelial expressed molecular pathways in these
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processes, especially the compromised epithelial P4 pathway rather than
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stromal. Beyond that, our findings provide valuable clinical reference that a
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better understanding of the uterine receptivity could advance discovery of
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novel biomarkers of uterine receptivity for diagnosing and treating infertility in
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human IVF-ET programs.
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ACCEPTED MANUSCRIPT Acknowledgments
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We are grateful to lab members Drs. Wengeng Lu and Zhaowei Tu for embryo
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transfer assistance. This work was supported by the National Key R&D Plan
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and National Basic Research Program of China (2017YFA0104603, 81601285
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to S.K.; 81130009, 81330017 and 81490744 to H. W.).
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Reference:
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[3] Cha J, Sun X, Dey SK. Mechanisms of implantation: strategies for successful pregnancy. Nat Med.18:1754-67.
[4] Paria BC, Huet-Hudson YM, Dey SK. Blastocyst's state of activity determines the "window" of implantation in the receptive mouse uterus. Proceedings of the National Academy of Sciences of the United States of America. 1993;90:10159-62.
[5] Paria BC, Reese J, Das SK, Dey SK. Deciphering the cross-talk of implantation: advances and
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[6] Red-Horse K, Zhou Y, Genbacev O, Prakobphol A, Foulk R, McMaster M, et al. Trophoblast differentiation during embryo implantation and formation of the maternal-fetal interface. J Clin Invest.
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[7] Cha J, Sun X, Dey SK. Mechanisms of implantation: strategies for successful pregnancy. Nat Med. 2012;18:1754-67.
[8] Zhang S, Lin H, Kong S, Wang S, Wang H, Armant DR. Physiological and molecular determinants of embryo implantation. Mol Aspects Med. 2013;34:939-80.
[9] Mulac-Jericevic B, Mullinax RA, DeMayo FJ, Lydon JP, Conneely OM. Subgroup of reproductive functions of progesterone mediated by progesterone receptor-B isoform. Science. 2000;289:1751-4. [10] Zhang S, Kong S, Wang B, Cheng X, Chen Y, Wu W, et al. Uterine Rbpj is required for
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embryonic-uterine orientation and decidual remodeling via Notch pathway-independent and -dependent mechanisms. Cell Res. 2014;24:925-42. [11] Song H, Han K, Lim H. Progesterone supplementation extends uterine receptivity for blastocyst implantation in mice. Reproduction. 2007;133:487-93. [12] Wang Q, Lu J, Zhang S, Wang S, Wang W, Wang B, et al. Wnt6 is essential for stromal cell
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proliferation during decidualization in mice. Biol Reprod. 2013;88:5. [13] Huang da W, Sherman BT, Lempicki RA. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc. 2009;4:44-57.
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[14] Ye X, Hama K, Contos JJ, Anliker B, Inoue A, Skinner MK, et al. LPA3-mediated lysophosphatidic acid signalling in embryo implantation and spacing. Nature. 2005;435:104-8. [15] Sun X, Terakawa J, Clevers H, Barker N, Daikoku T, Dey SK. Ovarian LGR5 is critical for successful pregnancy. FASEB J. 2014;28:2380-9. [16] Xiao S, Diao H, Zhao F, Li R, He N, Ye X. Differential gene expression profiling of mouse uterine luminal epithelium during periimplantation. Reprod Sci. 2014;21:351-62. [17] Jeong JW, Kwak I, Lee KY, Kim TH, Large MJ, Stewart CL, et al. Foxa2 is essential for mouse endometrial gland development and fertility. Biol Reprod. 2010;83:396-403. [18] Han BC, Xia HF, Sun J, Yang Y, Peng JP. Retinoic acid-metabolizing enzyme cytochrome P450 26a1 (cyp26a1) is essential for implantation: functional study of its role in early pregnancy. J Cell Physiol.
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ACCEPTED MANUSCRIPT 2010;223:471-9. [19] Wilcox AJ, Baird DD, Weinberg CR. Time of implantation of the conceptus and loss of pregnancy. N Engl J Med. 1999;340:1796-9. [20] Cooke PS, Spencer TE, Bartol FF, Hayashi K. Uterine glands: development, function and experimental model systems. Mol Hum Reprod. 2013;19:547-58. [21] Filant J, Spencer TE. Endometrial glands are essential for blastocyst implantation and decidualization in the mouse uterus. Biol Reprod. 2013;88:93.
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[22] Filant J, Spencer TE. Cell-specific transcriptional profiling reveals candidate mechanisms regulating development and function of uterine epithelia in mice. Biol Reprod. 2013;89:86.
[23] Diao H, Paria BC, Xiao S, Ye X. Temporal expression pattern of progesterone receptor in the uterine luminal epithelium suggests its requirement during early events of implantation. Fertil Steril. 2011;95:2087-93.
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Figure Legends:
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Figure1 Uterine receptivity and decidualization were defective beyond
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normal implantation "window"
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(A) Implantation status of blastocyst transferred on indicted days of PSP. (B)
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Post-implantation development of embryos transferred on indicted days of PSP.
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(C) The pregnancy outcome of day 4 blastocysts transferred into recipients on
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days 4 or 5 of PSP. Gross morphology (D) and weight induction (E) of uteri in
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response to artificial induction of decidualization in days 4, 5 and 6 PSP
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recipients. Data are shown as means ± SD, (*P<0.05, ***P<0.001).
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Figure2 Clustering analyses identified differentially expressed genes in
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days 4 and 5 PSP uteri
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(A) A volcano plot of gene expression in days 4 and 5 PSP uteri. (B) The Venn
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diagram showed the summary of total identified genes in days 4 and 5 uteri. (C)
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The heatmap of different expressed genes in each experimental group. The
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1-3 represented three independent experiments respectively. (D) Gene
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Ontology analysis of the 229 differentially expressed genes.
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ACCEPTED MANUSCRIPT Figure3 Differential expression of epithelium-expressing genes in days 4
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and 5 PSP uteri
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(A) The diagram showed the 107 different expressed ULE genes in the total
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229 differentially expressed genes. (B) Gene Ontology analysis of the 107
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different ULE genes. (C-F) mRNA expression level of Sprr2f, Foxa-2, Lpar3
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and Cyp26a1, were determined in days 4, 5 PSP uteri by quantitative
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Real-time PCR respectively. Values are expressed as mean ± SD from at least
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three independent samples (*P<0.05, **P<0.01).
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Figure4 Differential expression of epithelium specific P4-responsive
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genes in days 4 and 5 PSP uteri
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(A-H) Expression level of epithelial specific Fxyd4, Jam-2, Lrp-2, Areg, Galb1,
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Sox-17 genes and stromal special Hand-2, Hoxa-10 P4-response genes was
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determined in days 4, 5 PSP uteri by quantitative Real-time PCR, respectively.
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Values are expressed as mean ± SD from at least three independent samples
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(*P<0.05).
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ACCEPTED MANUSCRIPT Table 1. Implantation of blastocysts transferred into pseudopregnant mice No. of recipients with IS(%) 10(100) 8(100) 7(100)
No. of IS (%)
10 8 7
No. of embryos transferred 121 104 112
Day 5
24h 48h 72h
8 7 7
104 90 92
6(62.5) 7(100) 6(85.7)
31(29.8)* 35(38.9)* 36(39.1)*
Day 6
24h 48h 72h
5 5 5
69 66 74
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82(67.8) 81(77.9) 88(78.6)
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No. of recipients
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Day 4
Times after transfer 24h 48h 72h
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Days of Transfer
2(40) 1(20) 0
5(7.3) * 2(3.0) * 0*
ACCEPTED MANUSCRIPT Table 2. Functional categories of selected genes differentially expressed in day4 and day5 recipients according to gene ontology and KEGG pathway analyses Gene Symbol
Fold change
q-value(%)
Inflammatory response
Cxcl15 Kng1 Ccr2 Ccl21c Ccl21b C3 Pla2g7 Ccl8
4.09 0.26 0.48 0.42 0.42 0.42 0.47 0.45
0 0 0 0.3 0.3 0.55 0.55 0.9
Signal transduction
Tnfrsf21 Pex11a Iqgap2 Lpar3 Lgr5 Fga Arnt2 Ccr2 Ptger2 Tnfrsf11b Ms4a4c
2.02 2.16 2.00 2.15 0.11 0.05 0.38 0.48 0.31 0.46 0.28
0 0 0.23 0.43 0 0 0 0 0 0.61 0.74
Cxcl15 Pf4 Ccl21c Ccl21b Ccl8
4.09 0.44 0.43 0.43 0.46
0 0 0.30 0.30 0.99
Adh7 Mthfd2 Chdh Cdo1 Cbr2 Srd5a3 Cyp26a1 Me1 Xdh Gpx2
3.23 2.49 2.01 2.06 3.07 3.35 0.07 0.49 0.49 0.46
0 0 0 0 0 0.41 0 0 0.55 0.55
Oxidation reduction
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Chemotaxis
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Functional category
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ACCEPTED MANUSCRIPT The receptive state of the uterus is defined as a limited time period when the uterine milieu is favorable for blastocyst implantation. It is generally considered as day 4 of pregnancy in mice. In this study, by utilizing the embryo transfer
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model, we observed that a portion of blastocysts can initiate implantation even when transferred in day 5, while their mid-gestational development exhibited severe retardation, proving that the uterine status beyond the normal
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implantation window is unconducive for implantation. We therefore performed
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microarray analysis to explore the molecular basis that distinguishes the normal and defective uterine receptivity. Approximate 229 genes were found to be
differentially
expressed,
among
which
a
large
amount
were
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epithelial-expressing genes and those responsive to progesterone signaling.