A comparative analysis of gene expression induced by the embryo in the caprine endometrium

Abstract Transcriptomics is an established powerful tool to identify potential mRNAs and ncRNAs (non‐coding RNAs) for endometrial receptivity. In this study, the goat endometrium at estrus day 5 (ED5) and estrus day 15 (ED15) were selected to systematically analyse the differential expressed genes (DEGs) what were induced by the embryo. There were 1,847 genes which were significantly differential expressed in endometrium induced by the embryo at ED5, and 1,346 at ED15 (p‐value < .05). Secreted phosphoprotein 1 (SPP) was the responsive genes for embryo in the goat endometrium during estrus cycle, neurotensis (NTS) and pleiotrophin (PTN) were the responsive genes for embryo in the goat endometrium at ED5, Testin (TES) and Phosphate and Tension Homology Deleted on Chromsome ten (PTEN) at ED15. Furthermore, Gene Ontology (GO) and Kyoto Encyclopedia of Genes (KEGG) analysis revealed cytoplasm and Endocytosis were indispensable for the endometrium development in dairy goat. In a word, this resulting view of the transcriptome greatly uncovered the global trends in mRNAs expression induced by the embryo in the endometrium of dairy goats.

In our previous studies, the goat endometrium at estrus day 5 (ED5) and estrus day 15 (ED15) were selected to systematically analyse the transcriptome of endometrium With embryo using strand-specific Ribo-Zero RNA-Seq . The most important and studied factor in the implantation is the embryo-itself, however, there is no studies on the comparative analysis of mRNAs induced by the embryo in the endometrium of dairy goats. Then, we constructed a comprehensive analysis of the endometrial transcriptional profiles at the global level to compare the expressed mRNAs at ED5 and ED15, and further explore the differential expressed genes (DEGs). What's more, the Gene Ontology (GO) and Kyoto Encyclopedia of Genes (KEGG) were also analysed in the present study. Therefore, the results of the present study provided essential transcriptome to enhance the knowledge of mRNAs induced by the embryo in the endometrium of dairy goats.

| MATERIAL S AND ME THODS
All animals in this study were maintained according to the No. 5 proclamation of the Ministry of Agriculture, P. R. China, and the animal protocols were approved by the Review Committee for the Use of Animal Subjects of Northwest A&F University.

| Study design and sample collection
A total of 12 healthy, 24-month-old multiparous dairy goats (Xinong Saanen) were induced to oestrous synchronization for this study.
The protocols were applied to goats as follows: each goat was injected with 0.2 mg prostaglandin F2α (Ningbo Pharmaceutical Co., Ltd., China). On the same day (day 1), goats were administered intravaginal sponges containing 60 mg medroxyprogesterone acetate MPA). On day 10, each goat was twice injected with 20 IU of follicle-stimulating hormone (FSH) (Ningbo Pharmaceutical Co., Ltd.) (at 8.00 and 19.00 hr). On the day of sponge removal (day 11), each goat received an intramuscular injection of 0.1 mg prostaglandin F2α and 200 IU pregnant mare's serum gonadotropin (PMSG) (Ningbo Pharmaceutical Co., Ltd.). The experimental goats were observed three times daily to ascertain the estrous sign, and mated naturally twice in estrus (Lei et al., 2017;Liu et al., 2019;Zhang et al., 2015).
The 12 experimental goats were observed three times daily to ascertained estrous sign, and the first day of estrus was considered to be day 0 (ED0). Six goats mated naturally twice, and the other six unmated. The goats were euthanized following intravenous injection of a barbiturate (30 mg/kg) at day 5 (ED5, n = 6, 3 mated and 3 unmated) and day 15 (ED15, n = 6, 3 mated and 3 unmated), and the endometrium samples were obtained from the anterior wall of the uterine cavity. All samples were washed briefly with PBS (Phosphate Buffered Saline) and then immediately frozen in liquid nitrogen.

| RNA-seq date
Total RNA was extracted from endometrium using TRIzol reagent (Invitrogen, CA, USA), and the DNA contamination was evaluated using DNase (TaKaRa, Dalian, China) according to the manufacturer's instructions. The total RNA quantity and purity were analysed using the Bioanalyzer 2100 (Agilent, CA, USA) and RNA 6000 Nano LabChip Kit (Agilent, CA, USA) with RIN number > 7.0. Library construction, sequencing, reads mapping were showed in our previous studies (Lei et al., 2017;Liu et al., 2019).

| Differential expression of mRNAs in goat endometrium
Only comparisons with "q-values" less than 0.01 and statuses indicated as "OK" in the Cuffdiff output were regarded as showing differential expression. The fold-changes (log2 (ED15/ED5)) and corresponding significance threshold of the p-value were estimated according to the normalized mRNA expression levels. Based on the expression levels, the significant DECs between the ED15 and ED5 were identified using "p < .05" as the threshold.

| GO enrichment and KEGG pathway analysis
The hypergeometric test was applied to map all differentially expressed genes to terms in the Gene ontology (GO) database (ftp:// ftp.ncbi.nih.gov/gene/DATA/gene2 go.gz) (Consortium, 2004), which was an international standard gene functional classification system (Ashburner et al., 2000). The corrected p-value < .05 was used as the threshold to find significantly enriched GO terms in the input list of DEGs compared to their genomic background.
KEGG was the major public pathway-related database that helped to further understand the biological functions of genes with high level functions and the utilities of the biological system from large-scale molecular datasets (http://www.genome. jp/kegg/) . KEGG enrichment analysis identified significantly enriched metabolic pathways or signal transduction pathways (He & Liu, 2013) using the corrected P-value < 0.05 as a threshold to find significantly enriched KEGG terms in the input list of DEGs compared to The calculating formula of the corrected P-value:

| The common DEGs at ED5 and ED15 in the endometrium
In total, 2,906 DEGs were found, and 287 of these were co-expressed at both stages, 1,560 DEGs only were found at ED5, 1,059 DEGs were found at ED15 (Figure 1, Data S3). And then we pay more attention to the 1,059 DEGs at ED15, because these genes may be the responsive genes for the embryo. In other words, the expression levels of these 1,059 genes were regulated by embryo directly or indirectly in endometrium. Among these genes, TES and PTEN got our attention, because our previous studies showed that they play important roles in the endometrial cells during the development of endometrium in dairy goats .

| GO enrichment analysis
The DEGs were analysed by running queries for each DEG against the GO database, which provided information related to three ontologies: molecular function, cellular component and biological process.
At ED5, GO enrichments of the DEGs of ED5 were categorized into 663 functional groups that met the criteria of p-values < .05 (Data S4), and the distribution diagram was shown in Figure 2a.

| KEGG pathway analysis
Various genes cooperated with each other to exercise their bio- The KEGG analysis showed that map04144 (Endocytosis) was significantly enriched at both ED5, ED15 and common group suggesting that Endocytosis might participate in the goat developmental endometrium whether with embryo or not during estrus cycle.

| DISCUSS IONS
Many global transcriptomic studies of human endometrium have been published in the past years, providing considerable of omics studies in human endometrium and discovered that the number of genes identified in more than one study as potential biomarkers in endometrial physiology and pathophysiology has remained small, while any given study yields numerous candidate genes to explore.
In 2013, the domestic goat genome was generated using Whole Genome Mapping technology (Dong et al., 2013), which endowed its high quality of genome assembly and annotation. Currently, Ribo-Zero RNA-Seq is a competitive method to investigate the gene expression, particularly for incompletely annotated genomes (Derks et al., 2015;Sun et al., 2015). Our previous study reported the systematic identification and characterization of the developmental transcriptome landscape of the goat endometrium without embryo during estrus cycle using the Ribo-Zero RNA-Seq technology . What was of significantly higher depth, yielding 18 Gb of sequence for every sample, which was approximately seven times the size of the goat genome (2.66 Gb).

Some studies have reported modifications in gene expression
profiles associated with transition of the human endometrium from a pre-receptive to a receptive stage (Cuevas et al., 2016;Díaz-Gimeno et al., 2011;Hu et al., 2014a;Sigurgeirsson et al., 2017). However, only two genes were common to six of the seven largest studies, secreted phosphoprotein 1 (SPP1; previously known as osteopontin) (Wang & Yu, 2018). SPP1 is a glycoprotein involved in cellular adhesion and migration. It is the only factor identified that is common to most reported endometrial receptivity gene sets (Ruiz-Alonso et al., 2013), and it is reported to be an essential mediator of implantation and receptivity in humans (Gibson, Simitsidellis, Cousins, Critchley, & Saunders, 2016), what was confirmed in goats in this study.
Previously, much attention was devoted to the function of neurotensis (NTS) in cancer (Kontovounisios, Qiu, Rasheed, Darzi, & Tekkis, 2017), or as an important regulator of the fat absorption and obesity (Jing et al., 2016). NTS was detected in bovine endometrium, and the expression levels showed difference between the breeding and non-breeding seasons (Sakumoto et al., 2015).  Pufe, Bartscher, Petersen, Tillmann, & Mentlein, 2003). It is a secreted cytokines (Li et al., 1990) that plays roles in diverse biology process, such as cell adhesion, migration, survival, growth and differentiation (Deuel et al., 2002;Kadomatsu & Muramatsu, 2004; T. Muramatsu, 2002). Further study showed abnormalities on reproduction and development were observed in the PTN-KO mice (Muramatsu et al., 2006). What's more, PTN was stimulated by pregnancy, displayed a higher expression in the caruncular areas over the intercaruncular areas in bovine (Mansouriattia et al., 2009), and it increased in murine implantation sites during decidualization (Bany & Schultz, 2001). Considering the fact that PTN mainly expressed in the C areas of bovine endometrium, it may participate in the proliferation of stroma cells, and in the decidualization-like process (Johnson et al., 2003) in dairy goats. What's more, the protein levels of PTN increased with the increase in NTS in EECs . Considering the fact that PTN The changes in endometrial PTEN expression at both the mRNA and protein levels throughout the menstrual cycle were cyclic changes (Mutter, Lin, Kum, & Eng, 2000). Endometrial PTEN expression revealed temporal and spatial changes throughout the menstrual cycle, and during early pregnancy, E2 might down-regulate PTEN activity by increasing its phosphorylation. However, P4 was likely to regulate the PTEN pool by decreasing its phosphorylation and increasing its protein level (Guzeloglukayisli et al., 2003). Thus, we hypothesized that PTEN expression was also regulated by E2 and P4 in goat endometrium cells, and this conjecture was verified in the previous study in which we observed a direct regulation of PTEN by E2 and P4 in the EECs and ESCs of dairy goats .
TES is expressed in all normal human tissues (Sarti et al., 2005).
It is localized in the cytoplasm as a component of focal adhesions and cell-cell connections (Coutts, Mackenzie, Griffith, & Black, 2003;Griffith, Coutts, & Black, 2005). The protein encoded by the TES gene is a negative regulator of cell growth and may also function as a tumour suppressor (Steponaitis et al., 2016), as the loss of TES expression is frequently documented in various cancers (Z, 2014;Mcfarlane, 2001;Tatarelli, Linnenbach, Mimori, & Croce, 2000;Weeks, Ludgate, Lemée, & Morison, 2016;Zhong, Zhang, & Yin, 2015). In our previous study, it is observed that TES can promote EEC proliferation and inhibit cell apoptosis in vitro. TES could decrease BAX protein levels, and increase FAS in EECs in vitro, which is a critical factor for the progression of extrinsic apoptosis in human endometrum (Harada et al., 2004;Otsuki, 2001). Hence, we hypothesized that TES inhibited EECs apoptosis by decreasing the expression level of BCL-2/BAX via the MAPK pathway. Thus, modulation of TES expression in EECs may emerge as a potential target in regulating the development of pre-receptive endometrium in dairy goats.
In this study, TES and PTEN were up-regulated genes induced by the embryo in the endometrium of dairy goats at ED15 but not at ED5, and more in-depth research was needed to study the functions of NTS in EECs of in dairy goats.
During the implantation window, four endometrial cell types are identified in distinct proportions: microvilli-rich cells, pinopode cells, ciliated cells and others without apical differentiation. Zhioua highlighted important differences between surface and glandular epitheliums (Zhioua et al., 2012). Using transmission electron microscopy (TEM) for ultrastructural analysis, Zhioua showed images of endocytosis in epithelial cells of the endometrium, suggesting each cell type and each cell structure as a very precise function in order to prepare the endometrium to be receptive.
During the process of endocytosis cells engulf extracellular fluid (ECF) into plasma membrane invaginations which are then pinched off to form intracellular membrane-bounded vesicles, this is referred to as membrane mediated endocytosis (Quinn, Folkard, Detmar, & Casper, 2008). What's more, progesterone (P4) could control the endocytotic activity of the uterine epithelium and/or the movements of the endocytotic vesicles within the cells is supported by previous studies in the rat, as endocytosis is observed at Day 5 of pregnancy and during pseudopregnancy but not in cyclic animals (Vokaer & Leroy, 1962). Endocytosis occurred in pregnant and non-pregnant cows but was especially marked when circulating progesterone concentrations were high, and there was no evidence that pinopod-like functions could be attributed to large cytoplasmic protrusions from endometrial cells (Guillomot, Betteridge, Harvey, & Goff, 1986).
The GO and KEGG analysis showed that GO:0005737 (cytoplasm) and map04144 (Endocytosis) were significantly enriched with the most DEGs at both ED5, ED15 and common group, suggesting that cytoplasm and Endocytosis were indispensable for the goat developmental endometrium whether with embryo or not during estrus cycle.

| CON CLUS IONS
In this study, the goat endometrium at estrus day 5 and estrus day 15 were selected to systematically analyse the DEGs induced by the embryo in the endometrium of dairy goats. There were 1,847 genes that were differential expressed at ED5, and 1,346 at ED15.

| IMPLIC ATIONS
In this study, the goat endometrium at estrus day 5 and estrus day 15 were selected to systematically analyse the DEGs induced by the embryo in the endometrium of dairy goats. There were 1,847 genes that were differential expressed at ED5, and 1,346 at ED15. SPP1 was the responsive genes for embryo in the goat endometrium during estrus cycle, TES and PTEN were the responsive genes for embryo in the goat endometrium at ED15. Further GO and KEGG analysis revealed that GO:0005737 (cytoplasm) and map04144 (Endocytosis) were indispensable for the endometrium development in goat. In a word, this resulting view of the transcriptome greatly uncovered the global trends in mRNAs expression induced by the embryo in the endometrium of dairy goats.

ACK N OWLED G EM ENTS
The authors acknowledge XinLongMen dairy goat Breeding Farm for providing the goats.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no competing interests.

E TH I C A L S TATEM ENT
In this study, the Review Committee for the Use of Animal Subjects of Northwest A & F University approved the animal protocols. And all experimental dairy goats were maintained according to the No. 5 proclamation of the Ministry of Agriculture, P. R. China.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.