Preimplantation Endometrial Transcriptomics in Natural Conception Cycle of the Rhesus Monkey
Abstract
:1. Introduction
2. Materials and Methods
2.1. General Procedures
2.2. Animal Groups and Tissue Collection
2.3. RNA Extraction
2.4. Whole-Genome Expression Array
2.5. Array Data Analysis and Post-Hoc Enrichment Analysis
2.6. Quantification of Candidate Gene Expression by Real-Time RT-PCR
2.7. Immunohistochemical Localisation of Candidate Proteins for Orthogonal Validation
3. Results
3.1. General
3.2. Expression Array
3.2.1. Descriptive Data Analysis
3.2.2. Differentially Expressed (DE) Genes
3.2.3. Group-Specific Expression of Non-Matching Genes
3.3. Immunohistochemistrical Validation
4. Discussion
4.1. Genes Expressed in Both Groups and Involved in Protein Synthesis and Secretion along with Metabolic Processes Might Be Regulated by Progesterone
4.2. Genes Uniquely Expressed in Natural Conception Cycle and Involved in Induction of Immunotolerance to Non-Self Antigens Might Be Regulated by Factors in Seminal Plasma
- Immunotolerance to ovo-implantation in a natural pregnancy cycle is a primary process triggered in receptive endometrium, and it is not exclusively dependent on trophoblastic input, although it has—in all likelihood—reinforcing action to the process for successful establishment of pregnancy [52,53].
- The underlying genomic expression of the immunotolerance process in secretory phase endometrium is unlikely to be regulated by the preimplantation stage embryo because, in primates including the rhesus macaque, embryos seldom reach uterine lumen on Day 2 after ovulation [56]. Still, in the present study, we observed from transcriptomic expressions, endometrium geared towards immune tolerance to non-self antigens on Day 2 post-fertilization onwards.
- A plausible explanation for the observed differentiation towards immunotolerance in the secretory phase endometrium of mated and proven fecund group of animals could be due to the presence of seminal plasma inducing the observed genomic expression. In fact, evidence based on a series of elegant experiments is now available across various mammalian species to indicate that seminal plasma exerts substantial modifying action on endometrium towards receptivity, ovo-implantation, immunotolerance, embryo development, and establishment of pregnancy [38,52,57,58,59,60,61,62]. To our knowledge, this is the first report supporting the notion that seminal plasma could induce at the genomic expression level a process bias for immunotolerance to non-self antigens in the secretory phase endometrium of natural conception cycle in the rhesus macaque.
4.3. Genes Involved in Cell Polarity, Differentiation, Signaling, Fate, and Immunomodulation Might Be Regulated by Embryo-Derived Signals
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Group a (Subgroup) | Day after Ovulation b | Number of | ||||
---|---|---|---|---|---|---|
Animals Recruited | Ovulated | RNA Samples Obtained/Extracted c | Used in Array/Qrtpcr d | Tissue Samples Used Immunohistochemistry e | ||
1(A) | 2 | 12 | 10 | 5 | 4 | 4 |
1(B) | 4 | 12 | 11 | 5 | 4 | 4 |
1(C) | 6 | 11 | 10 | 5 | 4 | 4 |
Total | 35 | 31 | 15 | 12 | 12 | |
2(A) | 2 | 12 | 10 | 6 | 4 | 4 |
2(B) | 4 | 12 | 10 | 5 | 4 | 4 |
2(C) | 6 | 12 | 10 | 5 | 4 | 4 |
Total | 36 | 30 | 16 | 12 | 12 |
1. GEO accession number | GSE108760 | ||
2. Microarray details | |||
Product name: | Rhesus (V2) Macaque gene expression microarray | ||
Product format per slide: | 4 × 44 K; 60-mer printed oligonucleotides | ||
Total number of probes/chip: | 45,018 | ||
Total number of genes/chip: | 17,987 | ||
Content source: | RefSeq Release 37, UCSC mRNA, | ||
UniGene Build 13,ENSEMBL Release | |||
56, rheMac2 | |||
3. Descriptive details of microarray data | |||
Total number of hybridized a | |||
Probes: | 20,277 | ||
Genes: | 12,489 | ||
4. Details of hybridized probes (genes), Group-wise | |||
Group details | Number of samples arrayed | Quantitative description of hybridized a probes (genes) | |
Estimate b | Per cent | ||
Group 1(a) | 4 | 11,218 ± 376 (7922 ± 214) | 55.3 (63.4) |
Group 1(b) | 4 | 9574 ± 372 (7645 ± 85) | 47.2 (61.2) |
Group 1(c) | 4 | 12,603 ± 592 (7394 ± 216) | 62.2 (59.2) |
Group 2(a) | 4 | 11,386 ± 337 (8805 ± 261) | 56.2 (70.5) |
Group 2(b) | 4 | 11,230 ± 462 (8294 ± 298) | 55.4 (66.4) |
Group 2(c) | 4 | 11,684 ± 334 (8374 ± 260) | 57.6 (67.0) |
Serial Number | Gene Name (Gene Symbol) | Fold Change Based on (pFDR) | |
---|---|---|---|
Expression Arrays | RT-PCR | ||
Between Day 6 and Day 2 after ovulation in conception cycle group (Group 1c vs. Group 1a) | |||
Up-regulated | |||
1. | Cholinergic receptor, nicotinic, delta (CHRND) | 18.68 (0.046) | 3.80 (0.011) |
2. | Forkhead box D3 (FOXD3) | 5.91 (0.024) | 5.76 (0.006) |
3. | Gap Junction Protein, delta 4, 40.1 kDa (GJD4) | 3.79 (0.019) | 26.29 (0.006) |
4. | Mitogen-activated protein kinase 8 interacting protein 3 (MAPK8IP3) | 5.39 (0.020) | 3.99 (0.014) |
5. | Meckel syndrome, type 1 (MKS1) | 6.29 (0.023) | 3.98 (0.029) |
6. | Nucleoporin 50 kDa (NUP50) | 2.18 (0.021) | 2.39 (0.011) |
Down-regulated | |||
1. | Adenylate cyclase 5 (ADCY5) | 5.95 (0.028) | 4.13 (0.026) |
2. | Adiponectin receptor 1 (ADIPOR1) | 4.45 (0.018) | 2.12 (0.030) |
3. | Nicotinamide N-methyltransferase (NNMT) | 16.77 (0.046) | 3.08 (0.018) |
4. | Protein associated with topoisomerase II (PATL1) | 2.01 (0.001) | 2.65 (0.012) |
5. | Phosphatidylinositol glycan anchor biosynthesis, class V (PIGV) | 2.01 (0.003) | 3.86 (0.007) |
6. | Transforming growth factor, beta receptor II (TGFBR2) | 3.24 (0.009) | 2.16 (0.013) |
7. | TOX high mobility group box family member 2 (TOX2) | 18.92 (0.015) | 13.85 (0.015) |
8. | von Willebrand factor A domain containing 5B1 (VWA5B1) | 10.58 (0.003) | 19.48 (0.001) |
Between conception cycle and non-fecund cycle groups on Day 6 after ovulation (Group 1c vs. Group 2c) | |||
Up-regulated | |||
1. | Apoptosis, caspase activation inhibitor (AVEN) | 2.02 (0.011) | 17.20 (0.012) |
Down-regulated | |||
1. | Adenylate cyclase 5 (ADCY5) | 6.14 (0.011) | 18.00 (0.007) |
2. | Nicotinamide N-methyltransferase (NNMT) | 18.02 (0.015) | 4.74 (0.002) |
3. | TOX high mobility group box family member 2 (TOX2) | 20.54 (0.039) | 2.10 (0.012) |
4. | von Willebrand factor A domain containing 5B1 (VWA5B1) | 11.27 (0.036) | 52.00 (0.001) |
Group Number (Group Description)(Number of Genes Group Specifically Expressed) | GO Process (p-Value) | Number of Network Objects | |
---|---|---|---|
Total | Input Data | ||
Group 1 (Conception cycle) [809] | Positive regulation of tolerance induction to non-self antigen (2.4 × 10−31) | 25 | 23 a |
Group 2 (Non-fecund cycle) [1899] | Metabolic process 5.6 × 10−55 | 11,851 | 1275 a |
Groups 1 and 2 [370] | Protein targeting to membrane (1.1 × 10−21)/and endoplasmic reticulum (3.7 × 10−21) | 109/114 | 25 a |
Metabolic process 4.0 × 10−21 | 155 | 15 a |
Marker Proteins (Alias) | Physiological Functions | Reference |
---|---|---|
Adenylate cyclase 5 (ADCY5) | Class III adenylate cyclase gene family expressed as transmembrane protein catalyzing the formation of cAMP in response to G-protein signaling. Required for stromal cell decidualization in mammals. | Gellersen and Brosens [83] |
Apoptosis, caspase activation (AVEN) | Involved in cell fate regulation, stabilizes pro-survival protein BclxL, inhibits pro-apoptosis protein Apaf-1 function, exhibits cell cycle regulatory function. | Thandapani et al. [84] |
Cholinergic receptor nicotinic, delta (CHRND) | Family of pentameric acetylcholine-gated ion channels expressed in both neuronal, non-neuronal cells. Mediates signal transduction independent of generating ion currents in non-neuronal cells. Suppresses pro-inflammatory cytokines in the cholinergic anti-inflammatory pathway (CAP) in epithelial and endothelial cells. | Stokes et al. [85] |
Connexin 40.1 (GJD4) | Involved in formation of gap junctional intercellular of communication (GJIC) for direct intercellular transfer ions and small molecules, cAMP, and cGMP and propagation of inositol trisphosphate-dependent calcium waves, involved in growth control, differentiation, and apoptosis of normal cells. | Saez et al. [86] |
Foxhead box D3 (FOXD3) | Tumor suppressor. Member of forkhead box (FOX) family of transcription factors bearing helix-turn-helix DNA binding motif that interacts with DNA consensus sequences for nuclear localization and transcriptional regulation. Required for maintenance of cellular pluripotency and epigenetic priming of enhancers. | Krishnakumar et al. [87] |
Meckel syndrome1 (MKS1) | Structural protein required for ciliogenesis. Also involved in planar cell polarity of epithelium and directional cell migration. | Singh and Mlodzik [88] |
Nicotinamide-N-methyl (NNMT) | Catalyses S-adenosyl-L-methionine (SAM) to generate S-adenosyl-homocysteine (SAH). SAM donates methyl groups to substrates including histones. High NNMT expression results in hypomethylated histone proteins. | Ulanovskaya et al. [89] |
Transforming growth receptor 2 (TGFBR2) | Mediates pleiotropic functions of TGFb (beta). isoforms 1–3. Involved in implantation stage immune surveillance through functioning of endometrium and placenta including their immuno-modulatory/anti-inflammatory actions. | Jones et al. [64] |
TOX high mobility group, box family member 2 (TOX2) | Transcription factor belonging to TOX family domain, involved in differentiation and maturation of different cell types. | Vong et al. [90] |
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Bhat, M.A.; Khan, M.A.; Lalitkumar, P.G.L.; Poorasamy, J.; Sengupta, J.; Ghosh, D. Preimplantation Endometrial Transcriptomics in Natural Conception Cycle of the Rhesus Monkey. Reprod. Med. 2022, 3, 16-35. https://doi.org/10.3390/reprodmed3010003
Bhat MA, Khan MA, Lalitkumar PGL, Poorasamy J, Sengupta J, Ghosh D. Preimplantation Endometrial Transcriptomics in Natural Conception Cycle of the Rhesus Monkey. Reproductive Medicine. 2022; 3(1):16-35. https://doi.org/10.3390/reprodmed3010003
Chicago/Turabian StyleBhat, Muzaffer A., Meraj A. Khan, P.G.L. Lalitkumar, Jeevitha Poorasamy, Jayasree Sengupta, and Debabrata Ghosh. 2022. "Preimplantation Endometrial Transcriptomics in Natural Conception Cycle of the Rhesus Monkey" Reproductive Medicine 3, no. 1: 16-35. https://doi.org/10.3390/reprodmed3010003
APA StyleBhat, M. A., Khan, M. A., Lalitkumar, P. G. L., Poorasamy, J., Sengupta, J., & Ghosh, D. (2022). Preimplantation Endometrial Transcriptomics in Natural Conception Cycle of the Rhesus Monkey. Reproductive Medicine, 3(1), 16-35. https://doi.org/10.3390/reprodmed3010003